WO2024106737A1 - Pharmaceutical composition for treating cancer comprising foxm1 mutant or foxm1 shrna - Google Patents

Pharmaceutical composition for treating cancer comprising foxm1 mutant or foxm1 shrna Download PDF

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WO2024106737A1
WO2024106737A1 PCT/KR2023/014663 KR2023014663W WO2024106737A1 WO 2024106737 A1 WO2024106737 A1 WO 2024106737A1 KR 2023014663 W KR2023014663 W KR 2023014663W WO 2024106737 A1 WO2024106737 A1 WO 2024106737A1
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cancer
foxm1
cells
nucleic acid
expression
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Korean (ko)
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허용
장해란
임형신
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한양대학교 에리카산학협력단
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Priority claimed from KR1020220153798A external-priority patent/KR20240081504A/en
Priority claimed from KR1020220156930A external-priority patent/KR20240086714A/en
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Definitions

  • the present invention relates to a pharmaceutical composition for inhibiting cancer cell growth, invasiveness, and metastasis using a point mutation of FoxM1, a peptide containing a FoxM1 point mutation, or shRNA that specifically binds to FoxM1.
  • Cancer can be divided according to the stage of cancer progression, and in particular, the metastasis of cancer depending on the stage is an important criterion in determining treatment methods. Although the size of the cancer is important, treatment strategies should be reviewed by dividing it into primary cancer, which is the primary cancer, and metastatic cancer that moves to nearby lymph nodes or organs. As is well known, even if the primary cancer is treated, the survival rate is very low if metastasis cannot be prevented. Metastatic cancer varies depending on the type of cancer, but can account for up to 90% of deaths in cancer patients, so the prognosis is very poor (Dillekas, et al., Cancer Med 8 (2019) 5574-5576; Khan, I.
  • Macrophages which have the function of eliminating cancer, are also known to differentiate into tumor-associated macrophages (TAM) due to IL6, VEGFA, and TGFB1 secreted by cancer cells, and act as cells that help cancer survive (Murray) P. J. Physiol (2017) 79:541-566; J. Leukoc Biol (2007) 81 (2) 557-566; PLoS One (2016) 1):e0191040).
  • TAM tumor-associated macrophages
  • FoxM1 belongs to the large family of forkhead box (Fox) transcription factors and is a transcriptional regulator with a common DNA binding domain called 'wingled-helix' (Kaufmann, E. and Knoechel, W., Mech Dev 57 (1996) 3-20).
  • the FoxM1 transcription factor plays an essential role in the regulation of a wide range of biological processes, including cell proliferation, cell cycle progression, cell differentiation, DNA damage repair, tissue homeostasis, angiogenesis, and apoptosis. FoxM1's expression peaks in the S phase and G2/M phase of the cell cycle, and is known to play a key role in cell cycle progression (Laoukili, J. et al., Nat Cell Biol 7 (2005) 126- 136).
  • the mitotic transcription factor FoxM1 is a putative EMT regulator by activation of EMT transcription factors including SNAI1 and SNAI2. It can stimulate the expression of genes involved in various stages of tumor metastasis, including epithelial to mesenchymal-like transition, cell migration, and metastatic niche formation. Increased expression of FoxM1 is associated with liver cancer (YU, Chun-Peng et al. Molecular medicine reports 16.4 (2017) 5181-5188) and prostate cancer (Kalin, Tanya V.
  • colon cancer (Yoshida, Yuichi et al., Gastroenterology 132.4 (2007) 1420-1431), brain cancer (Liu, Mingguang et al., Cancer research 66.7 (2006) 3593-3602), breast cancer (Millour, Julie and E. W. Lam. Breast Cancer Research 12.1 (2010) 1-1), lung cancer (Wang, I-Ching et al. PLoS One 4.8 (2009): e6609), colon cancer, pancreatic cancer, skin cancer, cervical cancer, ovarian cancer, oral cancer, blood cancer, and nervous system. It is also observed in various malignant tumors, including (BARGER, Carter J.
  • FoxM1 increases in growing and dividing cells, and its expression and activity peak in the cell division phase of the cell cycle. Therefore, it is known to have a high expression rate in rapidly growing cancer cells (Liao, Guo-Bin et al., Cell Communication and Signaling 16.1 (2016): 1-15). Recently, it has been reported that the expression of FoxM1 increases depending on the stage in various carcinomas, suggesting that FoxM1 is related to cancer metastasis in addition to its function of leading growth.
  • colon cancer (Fei, BaoYing et al., Oncology Letters 14.6 (2017): 6553-6561), lung cancer (Wei, Ping et al., Int J Biol Sci 11.2 (2015): 186), and ovarian cancer (Chan, David W. et al., Oncogene 36.10 (2017): 1404-1416) reported that the expression increased as the stage increased (Li, Lijun et al., Oncotarget 8.19 (2017): 32298). Therefore, suppressing the expression of FoxM1, which is highly expressed in high-stage cancer, is expected to be highly effective in suppressing the growth of primary cancer and conversion to metastatic cancer.
  • Cancer metastasis is a phenomenon that occurs as a result of the primary cancer moving into an environment suitable for survival. If metastasis cannot be prevented, the patient's survival rate is very low due to cancer recurrence, etc., simply by removing the primary cancer. It is known that the rate of metastasis to nearby lymph nodes and organs increases as the size of the cancer increases.
  • Polo-like kinase 1 is a representative cell division factor that regulates cell growth, and its expression is high in various solid and blood cancers, so it is used in the diagnosis of various cancers. Recently, research results have reported that it can cause not only carcinogenesis but also metastasis (Shin et al., Oncogene (2020) 39(4) 767-785; Wu et al., Elife (2016) doi: 10.7554/eLife.10734.) It is being studied as a target molecule for metastasis. Based on these PLK1 characteristics, anticancer drug development research through the development of inhibitors for PLK1 is being competitively conducted by multinational companies (Yim, Anti-Cancer Drugs 24 (2013) 999-1006; Zhang, J. Med. Chem.
  • PLK1 has a kinase activity domain with an ATP-binding domain that can bind ATP and a polo box domain that binds substrates.
  • ATP-binding domain that can bind ATP
  • polo box domain that binds substrates.
  • phosphorylation occurs at the Thr 210 residue located in the phosphatase activity domain
  • activation of PLK1 is induced, which is an enzyme that phosphorylates Ser/Thr residues of substrate proteins bound to the Polobox domain (Barr et al., Nat Rev Mol Cell Biol 5 ( 2004) 429-440).
  • the expression of PLK1 increases during cell growth and division, and its activity is highest and peaks during cell division.
  • the cancer metastasis process is multi-step, and it is known that initial invasiveness and mobility are acquired through the EMT (Epithelial-mesenchymal transition) process of cancer cells (Dongre, Anushka, and Robert A. Weinberg. Nature reviews Molecular cell biology 20.2 (2019): 69 -84).
  • the EMT process is a process in which epithelial cells lose characteristics such as tight intercellular bonds and are converted into mesenchymal cells with migratory and invasive properties. At this time, looking at the changes in intracellular factors, the epithelial marker E-cadherin decreases while the mesenchymal markers N-cadherin, vimentin, SNAI1, and SNAI2 increase.
  • the present inventors developed a variant of the phosphorylation site of FoxM1 by PLK1 and made efforts to develop anticancer drugs based on gene and peptide therapy based on it.
  • the non-phosphorylated point mutant of Ser25 the PLK1 phosphorylation site of FoxM1
  • the phosphorylated point mutant at Ser25 of FoxM1 moves monocytes present in the tumor microenvironment to the periphery of the tumor, promoting differentiation into tumor-associated macrophages that help the tumor survive, and VEGFA, a key factor in angiogenesis, promotes differentiation.
  • the present inventors used FoxM1 shRNA, which suppresses FoxM1 expression, and thiostepton, a FoxM1 inhibitor, to inhibit the metastatic properties of lung cancer. , found that it significantly reduced the invasiveness, tumor formation, and differentiation of tumor-related macrophages, and found that FoxM1 shRNA and the FoxM1 inhibitor thiostepton can be useful in the treatment of metastatic cancer by blocking and reducing the metastasis, invasiveness, and tumor formation of various solid tumors. The present invention was completed by confirming that it exists.
  • the present invention provides that FoxM1 protein and peptide containing a point mutation, or shRNA that can specifically bind to FOXM1 and inhibit the expression of FOXM1, suppresses growth, invasiveness, and metastasis in solid tumors, and FoxM1 containing a non-phosphorylated point mutation Cancer using a strong inhibitory effect on the activity of immune cells that help the metastasis and invasiveness of cancer cells in lung cancer cells and immune evasion of tumors in the tumor microenvironment by proteins, fragments thereof, or shRNA that specifically binds to FOXM1. It is intended to provide a cure.
  • the present invention provides a pharmaceutical composition for treating cancer, comprising a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with a non-phosphorylated amino acid.
  • the non-phosphorylated amino acid is Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His, or Lys, and in another embodiment of the present invention
  • the cancer includes bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, and ovarian cancer.
  • rectal cancer cancer of the anal area, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of sexual and reproductive organs, Hodgkin's disease , esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal tumor, nerve glioma, meningioma, or pituitary adenoma, and in another embodiment of the present invention, the pharmaceutical composition inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
  • CNS central nervous system
  • a polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, 10 or more consecutive amino acids, and the 25th amino acid Ser is substituted with a non-phosphorylated amino acid.
  • a polypeptide is provided, wherein the polypeptide comprises an amino acid sequence represented by SEQ ID NO: 2, 4, or 5.
  • a polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, 10 or more consecutive amino acids, and the 25th amino acid Ser is substituted with a non-phosphorylated amino acid,
  • a pharmaceutical composition for treating cancer is provided.
  • the polypeptide includes an amino acid sequence represented by SEQ ID NO: 2, 4, or 5, and in another embodiment of the present invention, the cancer is bone cancer, blood cancer, lung cancer, small cell lung cancer, arsenic cancer, etc.
  • Cellular lung cancer squamous cell carcinoma, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, and prostate cancer.
  • uterine cancer endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer.
  • a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with a non-phosphorylated amino acid, or a polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, and 10 or more consecutive amino acids Provides a nucleic acid molecule encoding a polypeptide, or a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 4, or 5, wherein the 25th amino acid Ser is substituted with a non-phosphorylated amino acid, and other embodiments of the present invention
  • the 73rd to 75th nucleic acid of the nucleic acid sequence represented by SEQ ID NO: 3 is 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5' A nucleic acid molecule substituted with -GAG-3';
  • the 73rd to 75 is 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5' A
  • nucleic acid molecule that is substituted and comprises the 61st to 90th nucleic acid sequences;
  • the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'.
  • a nucleic acid molecule that is substituted and comprises the 70th to 99th nucleic acid sequences Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. is substituted and includes the 52nd to 81st nucleic acid sequences.
  • a recombinant vector containing the above nucleic acid molecule is provided.
  • a recombinant cell containing the above recombinant vector is provided.
  • the cancer metastasis process is determined to be highly likely. Provides an information provision method for determining the risk of metastasis.
  • the 73rd to 75th nucleic acid of the nucleic acid sequence represented by SEQ ID NO: 3 in the subject's cancer cells is 5'-GAT-3', 5'-GAC-3', 5'-GAA-3. ', or 5'-GAG-3', provides a method of providing information for determining the risk of cancer metastasis, including the step of determining that the possibility of cancer metastasis is high.
  • a recombinant metastatic cancer cell that expresses a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with Asp or Glu, which can be used to study metastatic cancer, is provided.
  • the 73rd to 75th nucleic acid of the nucleic acid sequence represented by SEQ ID NO: 3 is 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'.
  • recombinant metastatic cancer cells that can be used to study metastatic cancer, comprising nucleic acids substituted with '-GAG-3'.
  • the nucleic acid molecule comprises a nucleic acid sequence of any one of SEQ ID NOs: 7 to 10, and the nucleic acid molecule of the present invention
  • the nucleic acid molecule is any one selected from the group consisting of shRNA, siRNA, antisense RNA, antisense DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme
  • the The cancer is a cancer that overexpresses the FOXM1 protein or expresses a FOXM1 protein in which the 25th amino acid Ser is replaced with Asp or Glu.
  • any nucleic acid sequence comprising any one of SEQ ID NOs: 7 to 10 and selected from the group consisting of shRNA, siRNA, antisense RNA, antisense DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme
  • the nucleic acid molecule binds complementary to a gene or mRNA encoding FOXM1 protein, and in another embodiment of the present invention, the nucleic acid 187 to 207 or 709 to 709 of SEQ ID NO: 1 Binds specifically to nucleic acid number 729.
  • a recombinant viral vector comprising any one nucleic acid sequence of SEQ ID NOs: 7 to 10, and in another embodiment of the present invention, the recombinant viral vector is shRNA, siRNA, antisense RNA, antisense Expresses any one selected from the group consisting of DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme.
  • the pharmaceutical composition comprising administering a recombinant viral vector to a subject in need of cancer treatment.
  • the protein and/or peptide or shRNA containing the FoxM1 point mutation of the present invention can be usefully used in the treatment of various diseases caused by abnormal growth of cancer cells, especially cancer diseases such as primary and metastatic solid cancer and leukemia.
  • Figure 1 shows the results of analyzing the relationship between the expression of FoxM1 and PLK1 in adenocarcinoma patients among non-small cell lung cancer patients and patient survival rate.
  • A This is a graph analyzing the correlation between FoxM1 and PLK1 mRNA expression in lung cancer using big data from cBioPortal using Spearman and Pearson.
  • E This is a graph analyzing the survival rate of lung cancer patients with metastatic lung cancer according to the expression of PLK1 and FoxM1 through KM PLOTTER analysis.
  • F This is a graph analyzing the survival rate of lung cancer patients according to the stage of metastatic lung cancer according to the expression of PLK1 and FoxM1 through KM PLOTTER analysis.
  • G This is a graph showing the expression of epithelial-mesenchymal transition markers in lung cancer patients according to the stage of metastatic lung cancer through heatmap analysis.
  • Figure 2 shows the results of analyzing the clinical correlation between FoxM1 and active PLK1 in metastatic lung cancer cells.
  • A This is a graph showing the mRNA expression of FoxM1, PLK1, and epithelial-mesenchymal transition markers in lung cancer cells A549 treated with TGF- ⁇ using real-time polymerase chain reaction (Real-time PCR).
  • Figure 3 shows the results of analysis of phosphorylation and phosphorylation sites of FoxM1 by PLK1 active form.
  • Figure 4 is an experiment measuring the effect of overexpression of phosphorylated and non-phosphorylated point mutants of FoxM1 in lung cancer cell A549 on the mobility and invasiveness of cancer cells.
  • Figure 5 is an experiment measuring the metastatic and tumorigenic ability of cancer cells overexpressing phosphorylated and non-phosphorylated point mutants of FoxM1 in an animal model.
  • F Results of the degree of cell death by measuring the activity of caspase-3 enzyme in lung cancer cells expressing FoxM1 phosphorylation and non-phosphorylation point mutants.
  • Figure 6 shows the results of observing the effect on differentiation into tumor-associated macrophages (TAM) of monocyte THP-1 cells co-cultured with lung cancer cells expressing point mutants of the FoxM1 protein.
  • TAM tumor-associated macrophages
  • A Lung cancer cells expressing point mutants of the FoxM1 protein were co-cultured with monocyte THP-1 cells, and mRNA expression of M1 and M2 markers in THP-1 cells was measured using real-time polymerase chain reaction (Real-time PCR). This is the graph shown.
  • B Lung cancer cells expressing a point mutant of the FoxM1 protein were co-cultured with monocyte THP-1 cells, and M2-inducing factor and immune evasion factor mRNA expression was measured in A549 cells using real-time polymerase chain reaction (Real-time PCR). This is the graph shown.
  • Figure 7 shows the results of observing the effect on tumor cell immune evasion by differentiating monocyte THP-1 cells and Jurkat cells, a T cell, co-cultured with lung cancer cells expressing a point mutant of the FoxM1 protein.
  • A This is a graph showing the survival rate of lung cancer cells by co-culturing lung cancer cells expressing point mutants of the FoxM1 protein and monocyte THP-1 cells.
  • E Lung cancer cells expressing point mutants of the FoxM1 protein and Jurkat cells, which are T cells, were co-cultured in proportion, and the expression of T regulatory cell marker mRNA in Jurkat cells was shown using real-time polymerase chain reaction (Real-time PCR). It's a graph.
  • G This is a graph showing the survival rate of lung cancer cells by co-culturing lung cancer cells expressing point mutants of the FoxM1 protein, monocyte THP-1 cells, and Jurkat cells, which are T cells.
  • Figure 8 shows the results of observing the efficacy of the peptide by producing a FoxM1 non-phosphorylated point mutant peptide and evaluating its apoptosis effect and metastatic properties in lung cancer cells.
  • Figure 9 shows the results of analyzing patient survival according to FoxM1 expression in various carcinomas.
  • A This is a graph analyzing the survival rate of patients in 12 types of carcinoma according to the expression of FoxM1.
  • Figure 10 shows the results of producing a FoxM1 non-phosphorylated point mutant peptide and observing its effects on metastatic properties in lung cancer cells, tumor-related macrophage differentiation, and tumor cell immune evasion ability.
  • FITC a fluorescent substance
  • Figure 11 shows the results of analyzing the clinical correlation between FoxM1 and active PLK1 in metastatic lung cancer cells.
  • A This is a graph analyzing the related pathways of invasive cells in which active PLK1 is expressed in cancer metastasis conditions of lung cancer cell A549 through KEGG 2019 pathway analysis.
  • Figure 12 is an experiment measuring the effect of circular overexpression of FoxM1 by activated PLK1 on the mobility and invasiveness of cancer cells.
  • Figure 13 shows the results of observing the increase and differentiation of the TAM marker of THP-1 when co-culturing cancer cells expressing the original FoxM1 and THP-1 cells, which are human macrophages.
  • Figure 14 shows the results of observing the effect of suppressing FoxM1 expression when treated with each FoxM1 shRNA prepared to suppress FoxM1 mRNA expression in lung cancer cells.
  • A This is a graph showing the degree of inhibition of FoxM1 expression by treatment with each FoxM1 shRNA in lung cancer cell A549 in terms of mRNA expression pattern.
  • E This is a graph showing the mRNA expression pattern of epithelial-mesenchymal transition markers (CDH1, CDH2) in a cancer cell metastasis environment where suppression of FoxM1 expression by treatment of FoxM1 shRNA in lung cancer cell A549 is induced by TGF- ⁇ treatment.
  • CDH1, CDH2 epithelial-mesenchymal transition markers
  • Figure 15 shows the results of observing the differentiation ability of macrophages when A549 lung cancer cells expressing the FoxM1 circular protein were treated with FoxM1 shRNA, an inhibitor of FoxM1 mRNA expression.
  • This result shows the mRNA expression of FoxM1 when A549 lung cancer cells expressing the FoxM1 circular protein were treated with FoxM1 shRNA, an inhibitor of FoxM1 mRNA expression.
  • Figure 16 shows the results of observing changes in the mesenchymal transition marker macrophage M2-inducing factor when lung cancer cells are treated with Thiostrepton, a FoxM1 inhibitor.
  • This result shows the mRNA expression pattern of mesenchymal transition markers (CDH2, VIM, SNAI1, SNAI2) in a cancer cell metastasis environment where inhibition of FoxM1 function by treatment with Thiostrepton in lung cancer cell A549 is induced by overexpression of original FoxM1 (WT). .
  • Figures 17 to 19 show the nucleic acid sequence of the wild-type FOXM1 gene (SEQ ID NO: 3) and the amino acid sequence of the FoxM1 protein (SEQ ID NO: 1)
  • Figures 20 to 22 show the sequences of SEQ ID NOs: 1 to 11:
  • SEQ ID NO: 6 is human FOXM1B mRNA with accession number U74613
  • SEQ ID NO: 7 is the forward sequence of the first target according to an embodiment of the present invention
  • SEQ ID NO: 8 is the reverse sequence of the first target according to an embodiment of the present invention
  • SEQ ID NO: 9 is the present sequence.
  • SEQ ID NO: 10 is the reverse sequence of the second target according to an embodiment of the present invention
  • SEQ ID NO: 11 is the protein sequence of FOXM1 1B.
  • the inventor of the present invention confirmed that the expression of FoxM1 and PLK1 is high in adenocarcinoma patients among non-small cell lung cancer patients and has an inverse relationship with patient survival rate, so they can be used as diagnostic markers for prognosis (Figure 1).
  • the survival rate of patients with high PLK1 and FOXM1 expression in lung adenocarcinoma patients is higher than that of lung adenocarcinoma. It was confirmed that the survival rate was significantly lower than the low patient survival rate (Figure 1E).
  • Figure 1F the survival rate of patients with high PLK1 and FOXM1 expression was significantly lower than the survival rate of patients with low expression of PLK1 and FOXM1.
  • the inventors of the present invention confirmed the correlation with cancer metastasis because the expression of FoxM1 and PLK1 is high in an environment where cancer metastasis is induced in non-small cell lung cancer cells and is proportional to the increase in PLK1 activity and phosphorylation of FoxM1 ( Figure 2 ).
  • the inventor of the present invention identified phosphorylation of FoxM1 by activated PLK1 and a new phosphorylation site in metastatic lung cancer cells induced by TGF- ⁇ ( Figure 3)
  • Immunoprecipitation was performed to explore the interaction between PLK1 and FoxM1 in cancer metastasis conditions induced by TGF- ⁇ treatment.
  • PLK1 protein was precipitated with agarose beads and PLK1 antibody, and interacting proteins were analyzed through immunoblotting.
  • FoxM1 and PLK1 bind to each other under conditions in which Myc-tagged FoxM1 was expressed and treated with TGF- ⁇ ( Figure 3A).
  • Phosphorylation of FoxM1 is known to be a transcription factor that regulates the expression of various factors required for cell division in the cell cycle. Phosphorylation at the serine (S) site at positions 715 and 729 of FoxM1 by PLK1 has been reported, and phosphorylation at this site is known to promote cell division (FU, Zheng et al., Nature cell biology (FU) 2008) 10.9:1076-1082). No studies have been reported on whether point mutations in these regions block the metastasis, invasiveness, or tumor formation of cancer cells under conditions where metastatic properties are increased by activated PLK1.
  • PLK1 is a tumor phosphatase protein that is activated during the EMT process (Shin et al., Oncogene (2020) 39(4) 767-785). To determine whether FoxM1 is phosphorylated by PLK1 due to the interaction of the two proteins, a phosphatase reaction method was used. carried out.
  • the present invention provides an effect of promoting metastasis of cancer cells by a phosphorylated point mutant protein at a new phosphorylation site of FoxM1 by PLK1 and an inhibitory effect thereof by a non-phosphorylated point mutant protein of FoxM1 (FIG. 4).
  • the inventors of the present invention confirmed the invasiveness-promoting effect of cancer cells by a phosphorylated point mutant of FoxM1 and the invasiveness-inhibiting effect of cancer cells by a non-phosphorylated point mutant of FoxM1.
  • the invasiveness of the lung cancer cell group expressing the phosphorylated point mutant protein of FoxM1 was increased compared to the control group and the original FoxM1 protein.
  • the highest cancer cell invasiveness was observed in the S25E phosphorylation point mutant of FoxM1.
  • a decrease in invasiveness was observed in the lung cancer cell group expressing a non-phosphorylated point mutant protein of FoxM1. Therefore, in lung cancer cells, the invasiveness-promoting effect in cancer cells by a phosphorylated point mutant at the S25 residue of FoxM1 and the invasiveness-inhibiting effect of cancer cells by a non-phosphorylated point mutant of FoxM1 were observed.
  • a wound healing assay was performed to observe the mobility of cancer cells in A549 cells expressing each phosphorylated point mutant of FoxM1 (S25E, S361E, S715E) and three point simultaneous mutant (EEE) proteins.
  • Figures 4F, 4G Cell mobility was observed by measuring the healing gap between cells under a microscope at 0 h, 24 h, 48 h, and 72 h, respectively ( Figure 4F). And at 72 hours, the relative distance was displayed as a bar graph, with the control group set as 0 ( Figure 4G).
  • the mobility of the cell group expressing the phosphorylated point mutant (S25E) of the S25 residue of FoxM1 was found to be increased compared to the control group, which was similar to the positive control group of TGF- ⁇ treated cells (5 ng/ml). It was observed that the mobility of cancer cells expressing the phosphorylated point mutant (S361E) and phosphorylated point mutant (S717E) proteins showed no significant difference compared to the control group. In addition, the three point simultaneous mutant (EEE) had weaker mobility than the point mutant (S25E), but increased mobility was observed compared to the control group.
  • the present invention promotes the tumorigenicity and metastasis of cancer cells as primary cancer by phosphorylated point mutant protein at S25 residue, a new phosphorylation site of FoxM1 by PLK1, and its inhibition by non-phosphorylated point mutant protein of FoxM1. Provides effect. ( Figure 5)
  • mice were administered FoxM1 S25E and S25A. A549 cells expressing the mutants were injected through the tail vein. After rearing for 12 weeks, the animals were laparotomized to observe the degree of metastasis and tumor formation of cancer cells in the organs ( Figure 5A).
  • caspase-3 enzyme activity was measured (Figure 5F). The caspase-3 enzyme activity was found to be highest in cells in which the FoxM1 non-phosphorylated point mutant was overexpressed. This showed that overexpression of the FoxM1 non-phosphorylated point mutant induced and increased apoptosis.
  • the present invention provides the effect of promoting the recruitment of monocytes to cancer cells by a phosphorylated point mutant of FoxM1 and the effect of suppressing the differentiation of monocytes into tumor-related macrophages by a non-phosphorylated point mutant of FoxM1 (Figure 6).
  • M2d-TAM M2d-tumor-associated macrophages
  • S25E FoxM1 point phosphorylation mutant
  • CD68 a broad macrophage marker, and CD163, a tumor-related macrophage marker, in mouse lung tissue
  • CD68 was detected in mouse lung tissue injected with A549 cells overexpressing the FoxM1 phosphorylated point mutant (S25E).
  • Figures 6E, 6F it was confirmed that high expression of the CD163 marker was observed.
  • lung tissue injected with A549 cells overexpressing the non-phosphorylated point mutant (S25A) the expression of CD68 and CD163 markers was observed to be significantly low.
  • the expression of CD68 and CD163 proteins was observed to observe the increase in macrophages around the tumor using lung tissue lysate by immunoblotting.
  • the present invention provides an effect of promoting tumor immune evasion response by a phosphorylated point mutant of FoxM1 and an effect of suppressing immune evasion response by a non-phosphorylated point mutant of FoxM1 (FIG. 7).
  • Tumor-related macrophages are reported to increase the survival of cancer cells by helping them evade immunity (NOY, Roy and POLLARD, Jeffrey W., Immunity (2014) 41.1: 49-61). Based on this, the survival rate of lung cancer cells was analyzed by co-culturing A549 cells expressing FoxM1 phosphorylation and non-phosphorylation point mutants with mononuclear THP-1 cells, and the ratio of A549 cells: THP-1 cells was 1:0, 1:0. :2, 1:4, 1:6, the survival rate of A549 (A549 S25E ) cells expressing the FoxM1 S25E phosphorylation point mutant was observed to gradually increase (Figure 7A). On the other hand, the survival rate of A549 (A549S25A) cells expressing the FoxM1 S non-phosphorylated point mutant showed little change compared to the control group.
  • PD-1 mRNA (CD279), an immune evasion factor, was found in THP-1 monocytes co-cultured with A549 S25A cells. ) expression was observed to be the highest ( Figure 7B).
  • the mRNA (CD274) expression of the immune evasion factor PD-L1 (CD274) was significantly increased in cells expressing the phosphorylated point mutant (A549 S25E ).
  • A549 S25E cells may also affect the properties of T cells
  • the expression of CD25 and CD29 expressed on TILs in co-cultured Jurkat cells was conducted to examine the differentiation of A549 S25E cells into tumor-infiltrating T lymphocytes (TILs). was observed. As a result, it was observed that the expression of CD25 and CD29 was significantly increased in Jurkat cells co-cultured with A549 S25E cells.
  • Jurkat cells co-cultured with A549 S25A cells showed a significant inhibitory effect compared to Jurkat cells co-cultured with A549 (A549 S25E ) cells expressing the original or phosphorylated point mutant (Figure 7E).
  • A549 S25A cells had an inhibitory effect on the tumor immune evasion response caused by TAMs or TILs in the tumor microenvironment after co-culture with A549 cells overexpressing FoxM1 phosphorylation and non-phosphorylation point mutants with THP-1 cells or Jurkat cells. Changes in survival rate were observed through analysis ( Figure 7G). Therefore, the present invention provides the effect of suppressing tumor immune evasion response by a non-phosphorylated point mutant of FoxM1.
  • the present invention provides an inhibitory effect on cancer cell invasiveness and mobility and differentiation into tumor-associated macrophages (TAM) by a non-phosphorylated point mutant peptide of FoxM1 (FIG. 8).
  • CPP cell-penetrating peptide
  • Tat trans-activator of transcription
  • HAV-1 human immunodeficiency virus type-1
  • FoxM1 non-phosphorylated point mutant peptides 5 ⁇ M FoxM1 were administered in A549 cells under overexpression conditions. After treatment (-S25A peptide), changes in STAT1, VEGFA, c-fos, IL6, and CD274 mRNA were observed. We observed that STAT1, VEGFA, c-fos, IL6, and CD274 mRNA expression, which was increased by overexpression of FoxM1, was suppressed by peptide treatment ( Figure 10G). Therefore, it was confirmed that FoxM1 non-phosphorylated point mutant peptide had an inhibitory effect on tumor-related macrophage differentiation and tumor cell immune evasion ability.
  • the present invention provides an anticancer treatment agent for primary cancer as well as metastatic cancer, comprising a FoxM1 inhibitor or a FoxM1 protein and peptide containing a non-phosphorylation point mutation as an active ingredient.
  • the anticancer agent containing the protein and peptide containing a FoxM1 point mutation of the present invention as an active ingredient is used to treat various carcinomas with excessive expression of FoxM1, especially lung cancer (LIANG, Sheng-Kai, et al. Oncogene, 2021, 40.30: 4847-4858), Breast cancer (ZIEGLER, Yvonne, et al. NPJ breast cancer, 2019, 5.1: 1-11), liver cancer (YU, Chun-Peng, et al. Molecular medicine reports, 2017, 16.4: 5181-5188), prostate cancer (Kalin , Tanya V., et al. Cancer research 66.3 (2006): 1712-1720), colon cancer (Yoshida, Yuichi, et al.
  • the term “percent identity” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. Also, in the art, “identity” refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by correspondence between strings of said sequences. “Identity” and “similarity” are used in Computational Molecular Biology ((Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York ( 1993); Computer Analysis of Sequence Data, Part I (Griffin, A.
  • sequence analysis software such as the Megalign program from the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), with default parameters. It can be performed using the Clustal alignment method (Higgins et al., CABIOS.
  • “similarity” between two polypeptides is determined by comparing the amino acid sequences and conserved amino acid substitutions of the polypeptide to the sequence of the second polypeptide. Identity or homology to these sequences herein is defined by aligning the sequences and introducing gaps, if necessary, to achieve maximum percent homology and not considering any conservative substitutions as part of the sequence identity. It is defined as the percentage of amino acid residues in the candidate sequence that are identical to the known peptide. N-terminal, C-terminal or internal extensions, deletions or insertions in the peptide sequence will not be interpreted as affecting homology.
  • homology refers to the percentage of identity between portions of two polynucleotides or two polypeptides. Correspondence between sequences from one part to another can be determined by techniques known in the art. For example, homology can be determined by aligning sequence information and directly comparing sequence information between two polypeptide molecules using readily available computer programs. Alternatively, homology can be determined by hybridizing the polynucleotides under conditions that form a stable duplex between homologous regions, followed by cleavage using a single-strand specific nuclease and size determination of the cleaved fragments. .
  • sequence similarity in all its grammatical forms refers to the degree of identity or relatedness between nucleic acid or amino acid sequences that may or may not have a common evolutionary origin (Reeck et al., Cell 50:667 (1987) reference).
  • two DNA sequences are “substantially homologous” or “when about 50% (e.g., at least about 75%, 90%, or 95%) of the nucleotides match at least over a defined length of DNA sequence. “substantially similar.”
  • Substantially homologous sequences can be identified by comparing sequences using standard software available in sequence data banks or, for example, Southern hybridization experiments under stringent conditions as defined for the particular system. Defining appropriate hybridization conditions is within the skill of the art (see, e.g., Sambrook et al., 1989).
  • substantially similar refers to a nucleic acid fragment in which a change in one or more nucleotide bases results in the substitution of one or more amino acids but does not affect the functional properties of the protein encoded by said DNA sequence. . “Substantially similar” also refers to a nucleic acid fragment in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate changes in gene expression by antisense or co-suppression techniques. “Substantially similar” also refers to modifications of a nucleic acid fragment of the invention, such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. Accordingly, it is understood that the invention encompasses more than the specific sequences exemplified. Each of the proposed modifications is within the ordinary skill in the art as it determines retention of biological activity of the encoded product.
  • substantially similar sequences encompassed by the present invention are defined by .
  • Substantially similar nucleic acid fragments of the invention are nucleic acid fragments whose DNA sequences are at least about 70%, 80%, 90% or 95% identical to the DNA sequence of the nucleic acid fragment reported herein.
  • a “variant” of a polypeptide or protein refers to any analog, fragment, derivative or mutation derived from the polypeptide or protein and retaining at least one biological property of the polypeptide or protein.
  • Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequence of the structural gene encoding the protein, or may involve differential splicing or post-translational modifications. A skilled artisan can create variants with one or more amino acid substitutions, deletions, additions or substitutions.
  • variants include, inter alia: (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to a polypeptide or protein, (c) variants in which one or more of the amino acids is a substituent. and (d) variants in which a polypeptide or protein is fused with another polypeptide, such as serum albumin.
  • Conservative variants also refer to amino acid sequences with sequence changes that do not adversely affect the biological function of the protein.
  • a substitution, insertion, or deletion is said to have an adverse effect on a protein if the altered sequence interferes with or destroys the biological function associated with the protein.
  • the total charge, structure, or hydrophobicity-hydrophilicity of a protein can be altered without adversely affecting its biological activity.
  • the amino acid sequence can be altered, for example, to make the peptide more hydrophobic or hydrophilic without adversely affecting the biological activity of the protein. Techniques for obtaining such variants are known to those skilled in the art, including genetic (suppression, deletion, mutation, etc.), chemical and enzymatic techniques.
  • a “conservative amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain.
  • Families of amino acid residues with similar side chains have been defined within the art. These families include amino acids with basic chains (e.g., Lys, Arg, His), amino acids with acidic side chains (e.g., Asp, Glu), and amino acids with uncharged polar side chains (e.g., Gly, Asn, Gln, Ser, Thr, Tyr, Cys), amino acids with nonpolar side chains (e.g., Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), amino acids with beta-branched side chains (e.g., Thr, Val, Ile ) and amino acids with aromatic side chains (e.g., Tyr, Phe, His).
  • Proteins or polypeptides may undergo post-translational modification processes such as phosphorylation.
  • Protein phosphorylation is a reversible process and is catalyzed by protein kinases. In mammals, most phosphorylation occurs on Ser, Thr, or Tyr residues in proteins or amino acids.
  • the phosphorylated residue Ser in order to inhibit protein phosphorylation, is replaced with non-phosphorylated amino acids Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His. , or Lys, and in one embodiment of the present invention, it may be substituted with Gly, Ala, val, or Cys.
  • Ser is replaced with Ala.
  • DNA “coding sequence” refers to a double-stranded DNA sequence that encodes a polypeptide and can be transcribed and translated into a polypeptide in cells in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • “Appropriate regulatory sequence” refers to a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding sequence, which may be used to control transcription, RNA processing, or stability of the associated coding sequence. Affects translation. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures.
  • Coding sequences may include, but are not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is to be expressed in eukaryotic cells, the polyadenylation signal and transcription termination sequence will generally be located on the 3' side of the coding sequence.
  • the term "recombinant vector” refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express the gene insert.
  • plasmid refers to an extrachromosomal element that often carries genes that are not part of the cell's main machinery and usually exists in the form of a circular double-stranded DNA molecule. Such elements may be autonomously replicating sequences, genetic integration sequences, phage or nucleotide sequences, linear, circular or supercoiled single- or double-stranded DNA or RNA, from any source, where: Numerous nucleotide sequences are linked or recombined to form a unique construct that can introduce the promoter fragment and DNA sequence for the selected gene product into the cell along with the appropriate 3'untranslated sequence.
  • a plasmid includes an origin of replication that functions in a bacterial host cell (e.g., Escherichia coli), and a selectable marker for detection of the bacterial host cell containing the plasmid.
  • expression vector refers to a vector, plasmid, or carrier designed to express an inserted nucleic acid sequence and then transform a host.
  • the cloned gene i.e., the inserted nucleic acid sequence, is generally placed under the control of regulatory elements such as promoters, minimal promoters, enhancers, etc. There are numerous initiation control regions or promoters useful for directing expression of a nucleic acid in a desired host cell and are well known to those skilled in the art.
  • any promoter capable of driving the expression of these genes may include viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue-specific promoters, pathogenesis- or disease-related promoters, and development-specific promoters.
  • SV40 early (SV40) promoter region promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), E1A or major late promoter (MLP) of adenovirus (Ad) , human cytomegalovirus (HCMV) immediate early promoter, herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) ) promoter, glyceraldehyde-3-phosphate dehydrogenase (GSPDH) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin C (Ubc) promoter, albumin promoter, mouse metallothionein-L promoter and transcription.
  • LTR 3' long terminal repeat
  • RSV Rous sarcoma virus
  • MLP major late promoter
  • Ad adenovirus
  • HMV human cytomegal
  • Regulatory sequences in the regulatory region ubiquitous promoters (HPRT, vimentin, ⁇ -actin, tubulin, etc.), intermediate filaments (desmin, neurofilament, keratin, GFAP, etc.), treatment Shows tissue specificity, such as promoters of genes (such as MDR, CFTR, or factor VIII forms), pathogenesis- or disease-related promoters, and elastase I gene regulatory regions that are active in pancreatic acinar cells.
  • ubiquitous promoters HPRT, vimentin, ⁇ -actin, tubulin, etc.
  • intermediate filaments desmin, neurofilament, keratin, GFAP, etc.
  • treatment Shows tissue specificity, such as promoters of genes (such as MDR, CFTR, or factor VIII forms), pathogenesis- or disease-related promoters, and elastase I gene regulatory regions that are active in pancreatic acinar cells.
  • Promoters that have been used in transgenic animals; an insulin gene regulatory region active in pancreatic beta cells, an immunoglobulin gene regulatory region active in lymphoid cells, a mouse breast cancer virus regulatory region active in testicular, breast, lymphoid and macrophage cells; Albumin gene, Apo AI and Apo AII regulatory regions active in the liver, alpha-fetoprotein gene regulatory region active in the liver, alpha1-antitrypsin gene regulatory region active in the liver, beta-active in bone marrow cells.
  • Nucleic acid “nucleic acid molecule,” “oligonucleotide,” and “polynucleotide” are used interchangeably and refer to ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecule”) or a polymeric form of a phosphate ester of a deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxycytimine or deoxycytidine; “DNA molecule”) or phosphorothioate. (phosphorothioate) and any of its phosphoric acid ester analogues, such as thioesters.
  • nucleic acid molecule and especially DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Accordingly, the term includes double-stranded DNA found in linear or circular DNA molecules (e.g., restriction enzyme fragments), plasmids, supercoiled DNA, and chromosomes, among others.
  • sequence is presented herein in accordance with the general convention of presenting the sequence only in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand with the sequence matching the mRNA).
  • a “recombinant DNA molecule” is a DNA molecule that has undergone molecular biological manipulation.
  • DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.
  • transfection refers to the uptake of exogenous or heterologous RNA or DNA by a cell.
  • exogenous or foreign RNA or DNA is introduced into a cell, the cell is “transfected” by such RNA or DNA.
  • a cell is “transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA results in a phenotypic change.
  • the transforming RNA or DNA can be inserted (covalently linked) into the dyed DNA to construct the genome of the cell.
  • RNA transcription refers to the biological production of a product encoded by a coding sequence.
  • DNA sequence containing the coding sequence is transcribed to form messenger-RNA (mRNA).
  • mRNA messenger-RNA
  • the messenger RNA is then translated to form a polypeptide product with relevant biological activity.
  • the expression process may include additional processing steps for the RNA transcription product (e.g., splicing to remove introns), and/or post-translational processing of the polypeptide product.
  • the present invention provides host cells containing the vector of the present invention.
  • Host cells include prokaryotic (e.g., bacteria) and eukaryotic (e.g., fungi, yeast, animals, insects, plants) cells and can be any cell suitable for expression of the fusion protein.
  • Suitable prokaryotic host cells include E. coli (e.g., strains DH5, HB101, JM109, or W3110), Bacillus, Streptomyces, Salmonella, Serratia, and Pseudomonas. Including, but not limited to, species.
  • Suitable eukaryotic host cells include COS, CHO, HepG-2, CV-1, LLCMK2, 3T3, HeLa, RPMI8226, 293, BHK-21, Sf9, Saccharomyces, Pichia, Hansenula, Including, but not limited to, Kluyveromyces, Aspergillus, or Trichoderma species.
  • the peptides, proteins, polypeptides, nucleic acids, siRNAs, shRNAs, or miRNAs described herein and additional agents, e.g., immune checkpoint inhibitors, can be administered as pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment. It may be administered in the form of any suitable pharmaceutical composition, which may include an acceptable carrier and optionally one or more adjuvants, stabilizers, etc.
  • the pharmaceutical composition is for use in therapeutic or prophylactic treatment, e.g., treating or preventing a disease, such as a cancer disease, such as those described herein.
  • composition relates to a formulation comprising therapeutically effective substances, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients.
  • the pharmaceutical composition is useful for reducing the severity of, preventing, or treating a disease or disorder by administering the pharmaceutical composition to an individual.
  • Pharmaceutical compositions are also known in the art as pharmaceutical formulations.
  • pharmaceutical compositions include peptides, proteins, polypeptides, RNA, or RNA particles as described herein.
  • the pharmaceutical composition herein may contain one or more adjuvants or may be administered together with one or more adjuvants.
  • adjuvant refers to a compound that prolongs, enhances, or accelerates an immune response.
  • adjuvants include a heterogeneous group of compounds such as oil emulsions (eg Freund's adjuvant), mineral compounds (eg alum), bacterial products (eg pertussis toxin) or immune-stimulating complexes.
  • adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, and chemokines.
  • the cytokine may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFN ⁇ , IFN ⁇ , GM-CSF, LT-a.
  • Additional known adjuvants are aluminum hydroxide, Freund's adjuvant or oils such as Montanide® ISA51.
  • Other adjuvants suitable for use herein include lipopeptides such as Pam3Cys.
  • composition according to the present specification is generally applied as a “pharmaceutically effective amount” and as a “pharmaceutically acceptable formulation.”
  • pharmaceutically acceptable refers to the non-toxic nature of a substance that does not interact with the action of the active ingredients of the pharmaceutical composition.
  • the term “pharmaceutically effective amount” or “therapeutically effective amount” means the amount that, alone or in combination with additional administration, achieves the desired response or desired effect.
  • the desired response preferably means inhibition of progression of the disease. This includes slowing down the progression of the disease, and in particular stopping or reversing the progression of the disease.
  • the desired response in the treatment of a disease may also be delaying the onset or preventing the onset of the disease or condition.
  • the effective amount of the composition described herein will depend on the condition being treated, the severity of the disease, the individual characteristics of the patient, including age, physical condition, height and weight, the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and the like. It will be decided depending on factors. Accordingly, the dosage of the compositions described herein can be determined according to these various properties. If the response in the patient is not sufficient with the first dose, higher doses (or effectively higher doses achieved by other, more localized routes of administration) may be used.
  • compositions herein may include salts, buffers, preservatives, and optionally other therapeutic agents.
  • the pharmaceutical compositions herein include one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • Preservatives suitable for use in the pharmaceutical compositions herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
  • excipient refers to a substance that may be present in the pharmaceutical compositions herein but is not an active ingredient.
  • excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.
  • dilutes and/or thins means a substance that dilutes and/or thins. Additionally, the term “diluent” includes any one or more of fluids, liquids or solid suspensions and/or mixed media. Examples of suitable diluents include ethanol, glycerol, and water.
  • carrier refers to a substance, which may be natural, synthetic, organic, or inorganic, that is combined with the active ingredient to facilitate, enhance, or facilitate administration of the pharmaceutical composition.
  • a carrier can be one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to a subject.
  • Suitable carriers include, but are not limited to, sterile water, Ringer's, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, especially, biocompatible lactide polymers, lactide/glycols. ride copolymers or polyoxyethylene/polyoxy-propylene copolymers.
  • the pharmaceutical composition herein comprises isotonic saline solution.
  • compositions may be selected depending on the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly.
  • the pharmaceutical composition is formulated for topical or systemic administration.
  • Systemic administration may include enteral administration or parenteral administration, involving absorption through the gastrointestinal tract.
  • parenteral administration means administration by any means other than via the gastrointestinal tract, such as by intravenous injection.
  • the pharmaceutical composition is formulated for systemic administration.
  • systemic administration is by intravenous administration.
  • the FoxM1 variant described herein, fragment thereof, or nucleic acid encoding the same is administered systemically.
  • co-administration means administering several compounds or compositions to the same patient.
  • the various compounds or compositions may be administered simultaneously, essentially simultaneously, or sequentially.
  • the materials, compositions and methods described herein can be used to treat an individual with a disease, e.g., a disease characterized by the presence of diseased cells that express an antigen.
  • a disease e.g., a disease characterized by the presence of diseased cells that express an antigen.
  • a particularly preferred disease is cancer.
  • the antigen is derived from a virus
  • the materials, compositions and methods may be useful in the treatment of viral diseases caused by the virus.
  • the antigen is a tumor antigen
  • the materials, compositions and methods are useful in the treatment of a cancer disease, wherein cancer cells express the tumor antigen.
  • the materials, compositions and methods described herein can be used in the therapeutic or prophylactic treatment of a variety of diseases, wherein the support of and/or activation of immune effector cells as described herein is used to treat cancer and infectious diseases. It is advantageous for the same patient.
  • the materials, compositions and methods described herein are useful for the prophylactic and/or therapeutic treatment of diseases associated with antigens.
  • disease refers to an abnormal condition affecting an individual's body.
  • a disease is often interpreted as a medical condition associated with specific symptoms and signs.
  • the disease may be caused by factors originating from an external source, such as an infectious disease, or may be caused by an internal dysfunction, such as an autoimmune disease.
  • “disease” is used in a broader sense to refer to any condition that, upon contact with an individual, causes pain, dysfunction, suffering, social problems, death, or similar problems in the afflicted individual. In a broader sense, it sometimes includes injuries, disabilities, disabilities, syndromes, infections, isolated symptoms, aberrant acts and structural and functional atypical deformities, which in other contexts and for other purposes may be considered distinct categories. You can. Diseases generally affect individuals not only physically but also emotionally, as living with various diseases can change an individual's perspective on life and personality.
  • treatment means the management and care of an individual for the purpose of combating a condition such as a disease or disorder.
  • the term refers to alleviating symptoms or complications, delaying the progression of a disease, disorder or condition, alleviating or alleviating symptoms and complications, and/or curing or eliminating a disease, disorder or condition, as well as treating a disease, disorder or condition.
  • Prevention is intended to encompass the full spectrum of treatment for a given condition suffering from an individual, such as the administration of a therapeutically effective compound, wherein prevention refers to treatment of the individual for the purpose of combating the disease, condition or disorder. It will be understood as management and care, and includes the administration of an active compound to prevent the onset of symptoms or complications.
  • therapeutic treatment refers to any treatment that improves the health status of an individual and/or prolongs (increases) the lifespan of an individual.
  • the treatment may eliminate the disease in the individual, stop or slow the progression of the disease in the individual, inhibit or slow the progression of the disease in the individual, reduce the frequency or severity of symptoms in the individual, and/or treat the individual currently suffering from or previously suffering from the disease. There may be a reduction in recurrence.
  • prophylactic treatment refers to any treatment intended to prevent a disease from developing in an individual.
  • prophylactic treatment or “prophylactic treatment” are used interchangeably herein.
  • the terms “individual” and “entity” are used interchangeably herein. These terms refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates) that may be susceptible to or susceptible to a disease or disorder (e.g., cancer). However, the person may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms “individual” and “subject” do not refer to a specific age and therefore encompass adults, older adults, children, and newborns. In embodiments herein, “subject” or “individual” is “patient.”
  • patient refers to an individual or entity in need of treatment, specifically an individual or entity suffering from a disease.
  • the goal is to mount an immune response against diseased cells expressing the antigen, such as cancer cells expressing tumor antigens, to reduce the migratory, invasive, and proliferative properties of the tumor cells; and treating diseases such as cancer diseases involving cells expressing antigens such as tumor antigens.
  • diseased cells expressing the antigen such as cancer cells expressing tumor antigens
  • immune response refers to an integrated body response to an antigen or a cell expressing an antigen, and refers to a cellular and/or humoral immune response.
  • Cell-mediated immunity refers to cells characterized by the expression of an antigen, particularly by presenting the antigen with class I or class II MHC. This means that it includes a cellular response to The cellular response involves cells called T cells or T lymphocytes that act as either “helpers” or “killers.”
  • Helper T cells also referred to as CD4+ T cells
  • killer cells also referred to as cytotoxic T cells, cytolytic T cells, CD8 + T cells, or CTLs
  • induce [or inducing] an immune response can mean the absence of an immune response to a particular antigen prior to induction, or the absence of an immune response to a particular antigen prior to induction at a basal level. It can mean that it exists and is strengthened after induction. Accordingly, “induce [or induce] an immune response” includes “enhance [or enhance] an immune response.”
  • immunotherapy refers to the treatment of a disease or condition by inducing or enhancing an immune response.
  • immunotherapy includes antigen immunization or antigen vaccination.
  • immuno or “vaccination” refer to the process of administering an antigen to an individual for the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.
  • macrophage refers to a subgroup of phagocytes created by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines, or microbial products non-specifically perform phagocytosis, killing exogenous pathogens within the macrophages through hydrolytic and oxidative attacks and decomposing the pathogens. Peptides derived from cleaved proteins are presented on the cell surface of macrophages, where they can be recognized by T cells and interact directly with antibodies on the B cell surface, activating T and B cells and further immune responses. It can be stimulating. Macrophages belong to the class of antigen-presenting cells. In one embodiment, the macrophage is a splenic macrophage.
  • disease involving antigen refers to any disease in which an antigen is involved, eg, a disease characterized by the presence of an antigen.
  • a disease involving an antigen may be an infectious disease, a cancer disease, or a simple cancer.
  • the antigen may be a disease-related antigen, such as a tumor-related antigen, a viral antigen, or a bacterial antigen.
  • the disease involving the antigen is a disease involving cells expressing the antigen, preferably on the surface of the cell.
  • cancer disease refers to or refers to a pathological condition in an individual typically characterized by uncontrolled cell proliferation.
  • cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • examples of such cancers include bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, Uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, and carcinoma of the sexual and reproductive organs.
  • Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer Includes spinal tumors, gliomas, meningiomas, and pituitary adenomas.
  • the term “cancer” also includes cancer metastases.
  • the pharmaceutical composition is administered together with an immunotherapy agent.
  • immunotherapeutic agent means any agent that can be involved in activating a specific immune response and/or immune effector function(s). This specification contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies may achieve their therapeutic effects on cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting proliferation of tumor cells. . In certain embodiments, the antibody is a monoclonal antibody.
  • Monoclonal antibodies can induce cell death through antibody-dependent cell-mediated cytotoxicity (ADCC), or they can bind to complement proteins and induce direct cytotoxicity, known as complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • anti-cancer antibodies and potential antibody targets (in parentheses) include Abagovomab (CA-125), Abciximab (CD41) , Adecatumumab (atumumab) (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), Altumomab pentetate (CEA), Amatuximab (Amatuximab) (MORAb-009), Anatumomab mafenatox (TAG-72), Apolizumab (HLA-DR), Arcitumomab (CEA), Atezoli Atezolizumab (PD-L1), Bavitux
  • One embodiment of the present invention provides a method of providing information for diagnosing or determining the risk of cancer metastasis.
  • the method for providing information according to the present invention is to determine whether Ser25 is phosphorylated in the wild-type FoxM1 protein (SEQ ID NO: 1) or contains S25D or S25E mutations in cancer cells isolated from a subject with cancer, and that the subject with cancer has a possibility of cancer metastasis. 5'-GAT-3', 5'-GAC-3', or the 73rd to 75th nucleic acids in the wild-type FOXM1 gene (SEQ ID NO: 3) in cancer cells isolated from a subject with cancer.
  • recombinant metastatic cancer cells expressing a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with Asp or Glu, and/or 73rd to 75th of the nucleic acid sequence represented by SEQ ID NO: 3
  • recombinant metastatic cancer cells comprising a nucleic acid wherein the nucleic acid is substituted with 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'.
  • 5'-GAT-3', and 5'-GAC-3' are codons that code for Asp
  • 5'-GAA-3', and 5'-GAG-3' are codons that code for Glu.
  • the metastatic cancer cells have high mobility, invasiveness, and/or proliferative properties, they can be used to study metastatic cancer, or to create an in vivo model of metastatic cancer by inoculating them into animals.
  • the present invention provides a pharmaceutical composition for cancer treatment containing shRNA that specifically binds to FOXM1.
  • the inventors of the present invention observed an increase in the expression of FoxM1 simultaneously with N-cadherin, vimentin, SNAI1, and SNAI2, which are mesenchymal cell markers among EMT markers, by TGF- ⁇ treatment, so that the increase in FoxM1 in the process of cancer metastasis is a biomarker. It was confirmed that this can be done.
  • FoxM1 was identified along with mesenchymal cell markers (N-cadherin, vimentin, SNAI1, SNAI2) using immunoblotting in non-small cell lung cancer cell lines A549, NCI-H358, and NCI-H460 cells treated with TGF- ⁇ for 48 hours. As a result of observing the increase, it was observed that it increased consistently in all cells (Figure 11B).
  • THP-1 cells were co-cultured with A549 cells expressing FoxM1, and the tumor-related macrophages in THP-1 cells were co-cultured.
  • a study on the increase and differentiation of phagocyte markers was performed using RT-PCR ( Figure 13)
  • M2d-TAM M2d-tumor-associated macrophages
  • A549 cells expressing the FoxM1 circular protein After co-culturing A549 cells expressing the FoxM1 circular protein with THP-1, it was observed that M2-inducing factors IL4, IL6, IL10, VEGFA, and immune evasion factor CD274 were all increased in A549 cells. This shows that A549 cells expressing the FoxM1 circular protein differentiate THP-1 cells into M2d-TAM.
  • the inventor of the present invention confirmed the inhibitory effect on the mobility of cancer cells by FoxM1 shRNA, a FoxM1 mRNA inhibitor (FIG. 14).
  • pLKO-puro.1-hFoxM1 plasmid was created using the pLKO-puro.1 vector to create shRNA targeting the nucleotide sequences at positions 187-207 and 709-729 of the human FoxM1 mRNA sequence.
  • the following sequences can be used as target sequences for human FoxM1 shRNA production, and oligonucleotides with the following sequences can be used as primers for shRNA production.
  • the accession number for the human FoxM1 mRNA gene in Pubmed is U74613 and has 3326 bp.
  • anti-sense region 5'-CCTTTTCCTCCATCTCTTGCT-3'
  • the lentivirus for expressing FoxM1 shRNA was purified and concentrated, and then the virus was infected with A549 lung cancer cells, and the level of mRNA and protein expression was observed to confirm the effect of suppressing FoxM1 expression ( Figures 14A and 14B). .
  • shRNA targeting the nucleotide sequence at position 709-729 target #2, 709-729 bp
  • had the most excellent effect on suppressing FoxM1 expression Figures 14A, 14B).
  • Lentivirus for expressing FoxM1 shRNA provides an inhibitory effect on the metastasis of cancer cells.
  • the lentivirus for expressing FoxM1 shRNA was infected with lung cancer cells, A549, and an experiment was conducted on the effect of inhibiting the mobility of lung cancer cells (Figure 14C).
  • the present invention provides an inhibitory effect on the conversion of THP-1, a mononuclear cell in the tumor microenvironment, into tumor-related macrophages caused by overexpression of a FoxM1 variant by using FoxM1 shRNA, a FoxM1 mRNA suppressor (FIG. 15).
  • the present invention provides an inhibitory effect on the metastasis of cancer cells by thiostepton, a FoxM1 inhibitor.
  • thiostepton a FoxM1 inhibitor
  • thiostepton decreased the expression of CDH2, VIM, SANI1, and SNAI2, which are mesenchymal markers of lung cancer cells (Figure 16A).
  • thiostepton not only reduced the metastatic properties of the cancer cells themselves, but also reduced the expression of tumor-related macrophage-inducing factors IL6 and VEGFA, and also reduced the expression of CD274, an immune evasion factor (Figure 16B).
  • the present invention provides an anticancer treatment for not only primary cancer but also metastatic cancer, comprising FoxM1 shRNA or FoxM1 inhibitor as an active ingredient.
  • a pharmaceutical composition comprising one or more inhibitory nucleic acids capable of inhibiting the expression or activity of a protein expressed by FOXM1 nucleic acid is disclosed.
  • a cancer patient may be treated, for example, by administering a therapeutically effective amount of an inhibitory nucleic acid to inhibit the expression or activity of the protein encoded by the FOXM1 nucleic acid in tumor cells, such as the patient's cells.
  • the inhibitory nucleic acid described in the composition of the present invention is substantially complementary to the nucleic acid sequence of the target gene FOXM1.
  • the present invention relates to a method of treating a patient diagnosed with cancer or a disease characterized by expression of FOXM1, by administering an inhibitory nucleic acid capable of inhibiting the expression or activity of the protein encoded by the FOXM1 nucleic acid.
  • the symptoms associated with overexpression can be alleviated and the disease can be treated.
  • an effective amount refers to an amount that causes inhibition, prevention or treatment of cancer in tissues, animals, humans, etc. of a compound, pharmaceutical composition, or drug capable of inhibiting the expression or activity of FOXM1.
  • a pharmaceutically effective amount of a compound capable of inhibiting the expression or activity of FOXM1 can be delivered as a pharmaceutical composition.
  • the pharmaceutical composition may be a product containing a compound that inhibits the expression or activity of FOXM1, wherein the product contains a specific ingredient in a specific amount or, directly or indirectly, a combination of specific ingredients in a specific amount. It may be a product caused by
  • an inhibitory nucleic acid that inhibits the expression or activity of FOXM1 can bind to a portion of the FOXM1 nucleic acid and regulate the expression of the protein encoded by FOXM1.
  • Nucleic acids according to the invention may contain exact or partial mismatch sequences in the target region.
  • a number of nucleic acid molecules can modulate the expression or activity of the protein encoded by FOXM1.
  • the nucleic acid molecules include antisense RNA, shRNA, siRNA, miRNA, RNA aptamer, DNA aptamer, and decoy RNA. Each nucleic acid molecule can be used to inhibit the expression or activity of FOXM1.
  • the inhibitory nucleic acid comprises a double-stranded structure comprising about 15 to about 50 base pairs, for example 21 to 25 base pairs, and having a nucleic acid sequence that is identical or nearly identical to the target gene or RNA in the cell. It may be siRNA.
  • Antisense nucleic acids include, but are not limited to, morpholinos, 2'-O-methyl polynucleotides, DNA, or RNA.
  • RNA polymerase III transcribed DNA contains promoters such as the U6 promoter. These DNAs are transcribed to produce linear RNAs that can act as small hairpin RNAs or antisense RNAs that can act as siRNAs within cells.
  • Inhibitory nucleic acids that inhibit the expression or activity of FOXM1 may include ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. Additionally, the form of the nucleic acid may be single-stranded, double-stranded, triple-stranded, or quadruple-stranded.
  • Inhibitory nucleic acids can be produced chemically or biologically or expressed by plasmids or recombinant viral vectors.
  • Inhibitory nucleic acids according to the invention may be short hairpin RNA (shRNA).
  • shRNA can be synthesized in vitro or formed in vivo by transcription from an RNA polymerase III promoter. The preparation of these shRNAs and their use for gene cleavage in mammalian cells is described by Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 16:948-58; McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D.
  • RNA interference in adult mice Nature, 418:38-9; McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., Sharp P. A. (2002). Gene silencing using micro-RNA designed hairpins.
  • shRNAs can be manipulated within cells or within animals to continuously and stably suppress the desired gene. Those skilled in the art will understand that shRNA can be processed within cells to produce siRNA.
  • the shRNA comprises the sequence of any one of SEQ ID NOs: 7-10. In one embodiment, the shRNA includes the sequence of SEQ ID NO: 7 or 8, which can be used to inhibit the expression or activity of FOXM1 in vivo. In one embodiment, the shRNA comprises the sequence of SEQ ID NO: 9 or 10, which can be used to inhibit the expression or activity of FOXM1 in vivo.
  • the inhibitory nucleic acid can be designed and synthesized to form a duplex with the target RNA, including a non-complementary region (3 to 6 nucleotides long) between the complementary regions of 15 to 30 nucleotides in length.
  • the inhibitory nucleic acid capable of inhibiting the expression or activity of the nucleic acid encoded by FOXM1 can be 18 to 100 nucleotides in length, and in one embodiment, can be designed by one of ordinary skill in the art to be modified into the mature form.
  • the mature miRNA may be 19 to 30 nucleotides, 21 to 25 nucleotides, or 21, 22, 23, 24, or 25 nucleotides in length, wherein the miRNA precursor is 70 to 100 nucleotides in length. It may have a hairpin structure.
  • inhibitory nucleic acids described above may be RNA, DNA, or oligomers or polymers of both.
  • the term can include oligonucleotides having naturally occurring nucleobases, sugars, and backbones as well as non-natural moieties with functional similarities.
  • the inhibitory nucleic acid may comprise sufficient complementary nucleotide sequence to bind to FOXM1.
  • sufficient complementarity may be 12 to 25 nucleotides, 13 to 23 nucleotides, 14 to 23 nucleotides, or 15 to 23 nucleotides.
  • the nucleic acid molecules of the invention may be of virtually any length.
  • Inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoding FOXM1 are 20 to 100 nucleotides long, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31.
  • the inhibitory nucleic acid is selected from the group consisting of at least 17, at least 18, and at least 19 inhibitory nucleic acids if the stability of the target gene transcript in the presence of the inhibitory nucleic acid of the invention is reduced compared to stability in the absence of the inhibitory nucleic acid.
  • at least 20, at least 21, at least 22, or at least 23 have a sequence that is at least 90% homologous to the target transcript for a length of at least 20, at least 21, at least 22, or at least 23, and/or FOXM1 if the inhibitory nucleic acid binds to the target transcript under stringent conditions. is considered to be targeting.
  • Inhibitory nucleic acids can be produced biologically using expression vectors.
  • the inhibitory nucleic acid of the present invention can be synthesized in vivo on a cell basis or produced in vitro through chemical synthesis.
  • Inhibitory nucleic acids that inhibit the expression or activity of the protein encoded by FOXM1 can be produced using chemical synthesis and/or enzymatic ligation reactions according to methods known in the art.
  • the inhibitory nucleic acid contains a region sufficiently complementary to the target nucleic acid and is of sufficient length so that the miRNA forms a duplex structure with the target nucleic acid.
  • Inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoded by FOXM1 include a region that is partially or fully complementary to the target RNA.
  • the inhibitory nucleic acid capable of inhibiting the expression or activity of the protein encoded by FOXM1 and the target sequence do not have to be completely complementary, but must be compatible enough to inhibit expression of the target gene.
  • Inhibitory nucleic acids can be synthesized and modified to have the desired properties. For example, modifications may be made to improve stability, increase the probability of thermodynamic hybridization with the target nucleic acid, increase targetability to a specific tissue or cell type, or increase permeability to cells. Modifications may be performed to increase sequence specificity and reduce non-specific binding.
  • the inhibitory nucleic acid molecule may have a nucleotide sequence that is substantially identical to a portion of the nucleic acid encoding FOXM1.
  • single-stranded oligonucleotides that are substantially identical to at least a portion of the nucleic acid encoding FOXM1 can be administered to patients who have cancer or are at risk of having cancer.
  • Inhibitory nucleic acids include naked plasmids or other DNA formulated in liposomes or expression vectors, including viral vectors, including DNA viruses and RNA viruses, including adenoviruses, lentiviruses, alphaviruses, and adeno-associated viruses. It can be delivered to cells in any of a variety of forms. The amount of nucleic acid needed can be determined by a person skilled in the art depending on the delivery formulation and whether the nucleic acid used is DNA or RNA.
  • the inhibitory nucleic acid capable of inhibiting the expression or activity of the protein encoding FOXM1 may be expressed from a transcription unit inserted into a DNA or RNA vector.
  • Recombinant vectors may be DNA plasmids or viral vectors.
  • Viral vectors suitable for producing inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoding FOXM1 can be designed, for example, based on adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. However, it is not limited to this.
  • Recombinant vectors capable of expressing inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoding FOXM1 can be delivered according to the above methods and can persist in target cells or provide only transient expression of the nucleic acid molecule. there is. These vectors can be administered repeatedly as needed. Once expressed, the inhibitory nucleic acid interacts with the target RNA and inhibits miRNA activity.
  • Numerous viruses can be used in the present invention, including papovaviruses, such as SV40, adenovirus, vaccinia virus, adeno-associated virus, herpesvirus, and retroviruses of avian, rodent, and human origin. there is.
  • lentiviral vectors may also be used in the present invention.
  • the lentiviral vector may be a doxycycline-inducible lentiviral vector engineered to express one or more shRNAs against FOXM1.
  • Delivery of a vector expressing an inhibitory nucleic acid can be administered systemically, such as by intravascular or intramuscular injection, or locally to a target organ or tissue.
  • nucleic acid hydrolases that cleave phosphodiester bonds in DNA are expressed in most cells, unmodified DNA, such as inhibitory oligonucleotides, is usually modified to avoid degradation. Additionally, since most targets of antisense exist within cells, the entry of nucleic acids into cells must be considered. For clinical use, inhibitory nucleic acids with nucleotides modified to prevent degradation are preferred. In addition, other molecules may be fused to increase selectivity for targeting specific cells or to enable them to pass through cell membranes.
  • Physiological activity can be significantly improved through chemical modification of inhibitory nucleic acids.
  • Antisense nucleic acids can be modified with phosphorothioate to improve binding ability to cell surface proteins.
  • Cell delivery can be improved by fusing positively charged arginine-rich peptides to PMO-modified antisense nucleic acids.
  • Intracellular delivery systems include antisense nucleic acid fusions and cationic lipid carriers, carrier molecules that bind to cell-specific receptors, cyclodextrins, dendrimers, microparticles, and macromolecules. This delivery system can improve intracellular delivery efficiency by protecting antisense nucleic acids from nucleic acid degradation and/or promoting absorptive endocytosis.
  • Macromolecules include cell-penetrating peptides (CPPs), short cationic peptide sequences fused to antisense nucleic acids through disulfide bonds.
  • CPPs include cell-penetrating peptides (CPPs), short cationic peptide sequences fused to antisense nucleic acids through disulfide bonds.
  • Commonly used CPPs include penetratin, HIV TAT peptide 48-60, and transportan. Additionally, by binding dioleylphosphatidylethanolamine to the liposome delivery system, it is possible to destabilize the endosomal membrane and promote the release of antisense nucleic acids after endocytosis.
  • nanoparticle-based siRNA delivery systems have been approved by the FDA and have been introduced into clinical trials for cancer treatment. All nanoparticle-formulated siRNA delivery systems currently in clinical trials for cancer treatment are based on polymers or liposomes.
  • nanoparticles fused to targeting ligands have an increased probability of binding to tumor surface receptors, but this process also increases the overall size of the nanoparticles.
  • Coating the nanoparticles with PEG reduces uptake by RES and consequently increases the circulating half-life, but reduces target specificity because the PEG molecules interfere with spatially selective binding. Therefore, it is important to select appropriate cell-specific targeting moieties and design stable and efficacious nanoparticle delivery systems. It is known that various nanoparticle-based delivery systems, such as cationic lipids, polymers, dendrimers, and inorganic nanoparticles, can effectively deliver siRNA in vitro and in vivo.
  • Antisense nucleic acids can be formulated in saline solution with chemical modifications to enable absorption and delivered systemically, such as by oral administration, or through topical administration to tumors.
  • Their phosphorothiate backbone binds to serum proteins and slows secretion by the kidneys.
  • the aromatic nucleobases interact with other hydrophobic molecules in serum and on the cell surface.
  • Many cell types express surface receptors that take up oligonucleotides in vivo, but these often disappear in cultured cells, suggesting that lipids may be more important for allele-specific oligonucleotide (ASO) delivery in culture than in vivo.
  • ASO allele-specific oligonucleotide
  • siRNA double-stranded nucleic acid
  • all aromatic nucleobases are located on the inside, and only the significantly hydrated phosphates are located on the outside of the double strand.
  • the hydrated surface has little interaction with the cell surface and is quickly excreted in the urine. Therefore, researchers have conducted a lot of research to develop siRNA delivery vehicles.
  • the main technique for delivering siRNA is mixing RNA with cationic and neutral lipids, although desired results have also been obtained with peptide transduction domains and cationic polymers.
  • PEGylated lipids By including PEGylated lipids in the formulation, the circulating half-life of the particles can be extended.
  • One way to optimize single-stranded DNA or RNA is to use chemical modifications to increase resistance to nucleases, such as introducing phosphorothioate (PS) bonds at the phosphodiester bond sites.
  • PS phosphorothioate
  • Such modifications greatly improve stability against digestion by nucleolytic enzymes.
  • PS binding also improves binding to serum proteins in vivo, increases half-sensitivity, and allows better delivery of the active ingredient to tissues.
  • ASOs containing only PS modifications can produce an antisense effect intracellularly, but the potency is not always high and the results are not routine enough to be reliable.
  • double-stranded siRNA has been widely utilized as a means to suppress gene expression.
  • double-stranded RNA enters the cell, it binds to the protein machinery of RISC.
  • the synthesized RNA used to delete genes is generally double stranded, 19 to 22 bp. This length is sufficient to form a stable duplex to be recognized by RISC, yet short enough to avoid the strong interferon response elicited by duplexes longer than 30 pb.
  • siRNA Since the first paper on gene deletion in mammalian cells was published in 2001, siRNA has been the subject of thousands of experimental studies to test its function. While antisense oligonucleotides continue to be used for gene deletion, the powerful properties of siRNA and the relative ease of identifying active siRNA have made them the preferred method of deletion in many laboratories.
  • unmodified double-stranded RNA is surprisingly stable, and chemically modified siRNA is generally not essential for defects in gene expression.
  • unmodified siRNA does not have high activity, and its properties can be significantly improved through chemical modification.
  • Chemically modified siRNAs can show improved nuclease stability and increased duration of activity. Unmodified RNA is also rapidly cleared, and chemical modification, complex formation with carriers, and localized delivery to disease targets can help achieve improved in vivo outcomes.
  • Oligonucleotides used in the present invention can be produced through commonly known techniques, for example, solid phase synthesis.
  • the equipment used in this synthesis method is commercially available, and other means may also be added for synthesis.
  • Therapeutic administration of inhibitory nucleic acids to cells to inhibit the expression or activity of proteins encoded by FOXM1 nucleic acids includes administering any nucleic acid known to those skilled in the art. To treat cancer, it can be delivered via oral administration or injection, or both. Nucleic acid molecules can be delivered to cells through a variety of methods known in the art, such as iontophoresis or transfer to other carriers such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioconjugate microspheres. Incorporation, or encapsulation into liposomes by proteinaceous vectors can be used.
  • the inhibitory nucleic acid to inhibit the expression or activity of the protein encoded by the FOXM1 nucleic acid is administered to a cell organelle, cell, tissue, tumor, or organism by subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal injection, etc. may be administered.
  • the above inhibitory nucleic acids or other active ingredients may be added to pharmaceutical compositions suitable for administration.
  • the pharmaceutical composition includes one or more inhibitory nucleic acids capable of reducing the expression or activity of the protein encoded by the FOXM1 nucleic acid and a pharmaceutically acceptable carrier.
  • the inhibitory nucleic acid may be provided as a sustained-release composition. Immediate or sustained release compositions may be determined depending on the condition of the patient being treated. If the patient's condition is an acute or hyperacute disease, the treatment should use an immediate-release composition, whereas in the case of preventive or long-term treatment, a sustained-release composition is suitable.
  • the anticancer agent containing FoxM1 shRNA or FoxM1 inhibitor or FoxM1 protein containing a point mutation of the present invention as an active ingredient especially various carcinomas and lung cancer with excessive expression of FoxM1 (Wang, I-Ching et al. PLoS One 4.8 (2009) : e6609), colon cancer (Yoshida, Yuichi et al., Gastroenterology 132.4 (2007) 1420-1431), prostate cancer (Kalin, Tanya V. et al., Cancer research 66.3 (2006) 1712-1720), breast cancer (Millour , Julie and E. W. Lam.
  • Example 1.1 Clinical correlation between FoxM1 and active PLK1 in metastatic lung cancer cells and expression analysis of FoxM1 and active PLK1 in cancer metastasis conditions induced by TGF- ⁇
  • Non-small cell lung cancer is divided into adenocarcinoma and squamous lung cell carcinoma.
  • Example 1.2 Phosphorylation of FoxM1 by activated PLK1 and identification of new phosphorylation sites in metastatic lung cancer cells induced by TGF- ⁇
  • the mRNA expression level of CDH2, SNA11, and SNAI2, mesenchymal markers, and the mRNA expression level of CDH1, an epithelial marker were increased. A decrease was observed. Under these conditions, an increase in the mRNA expression levels of PLK1 and FOXM1 was observed compared to the control group ( Figures 2A, 2B, 2C). In addition, the protein amounts of vimentin, PLK1, E-cadherin, and N-cadherin showed the same results as the mRNA expression levels.
  • the present inventors cut pLVX-TRE3G-eRFP with NotI and EcoRI, and then added the original FoxM1 (WT) plasmid to 5' -ACGGGGCCCATGAAAACTAGCCCCCGTCG-3' (forward primer). It was amplified by PCR using the primers 5'-ACGGGAATTCCTACTGTAGCTCAGGAATAA-3' (reverse primer), cut with NotI and EcoRI, and subcloned into vector pLVX-TRE3G-eRFP.
  • pCMV-VSV.G pCMV- ⁇ 8.2, pLVX-TRE3G-eRFP-Target or pLVX-Tet3G DNA was transfected to express lentivirus in HEK293 cells, and then viruses were collected. The intention was to use this on cancer cells. After transfection, the virus culture was collected at 12-hour intervals for up to 72 hours, filtered with a 0.2 mm filter, and centrifuged at 18000 rpm at 4°C for 90 minutes. The supernatant was discarded, and the viruses collected in TNE buffer were stored at 4°C and used to infect cancer cells from the next day.
  • Example 1.4 Evaluation of the effect of epithelial-mesenchymal transition after selection of cells expressing active and inactive genes of FoxM1 using lentivirus
  • lung cancer cells were cultured as follows to infect lung cancer cells with lentivirus expressing phosphorylated and non-phosphorylated point mutants of FoxM1.
  • the infected cells were first infected with pLVX-Tet3G-expressing lentivirus and treated with G418 for 5 days to select infected cells. .
  • the selected A549Tet3G cells were infected with lentivirus expressing the original and phosphorylated point mutant forms (S25E, S361E, S715E) and non-phosphorylated point mutant forms (S25A, S361A, S715A) of FoxM1, and then treated with puromycin for 48 hours.
  • a stabilized cell line was constructed by treatment for a period of time.
  • a migration assay was performed to observe the effect of these point mutants on the mobility of cancer cells in lung cancer cells A549 expressing the original, phosphorylated, and non-phosphorylated FoxM1 point mutant proteins.
  • Matrigel was completely dissolved at 4°C for 16 to 20 hours, and then Matrigel was diluted to 1 mg/ml with cold serum-free RPMI 1640 (4°C). 100 ⁇ l of Matrigel mixture (1 mg/ml) was added to an 8.0 mm 24 well insert and hardened in a 37°C incubator for 12-20 hours. Lung cancer cells A549 expressing each FoxM1 original, phosphorylated and non-phosphorylated point mutant protein were diluted in serum free RPMI 1640 (36°C) at a cell number of 1X10 5 cells/well and dispensed onto the hardened Matrigel insert. Here, 0.5 ml/well of warm RPMI 1640 (10% FBS) at 36°C was dispensed.
  • 0.5 ml of 36°C RPMI 1640 (10% FBS) containing 5 ng/ml TGF- ⁇ was used. After that, the medium was changed once every three days and the degree of invasion was observed. On the 7th day, when it was observed that cancer cell invasion had sufficiently occurred, the medium was removed, washed with 1XPBS, and the cells inside the insert were scraped off with a cotton swab. and washed with 1XPBS to remove any remaining cells and matrigel inside the insert.
  • cells expressing the FoxM1 point mutant gene by the lantivirus system are distributed in a 6-well plate at 1X10 5 cells/well. 24 hours after dispensing, scratches are made at regular intervals using a 200 ⁇ l pipette tip. Additionally, A549 cells treated with 5 ng/ml of TGF- ⁇ were used as negative control (Mock) and positive control. Observe at intervals of 0h, 24h, 48h, and 72h and measure the healing distance (Figure 4F). Additionally, the control group was converted to 0% at 72 h, and the relative distance was displayed as a bar graph ( Figure 4G).
  • the three point simultaneous mutant (EEE) was observed to have weaker mobility compared to the point mutant (S25E) at 15% at 72h, but increased compared to the control group. Therefore, it was found that the mobility of cancer cells was increased in the cell group expressing the phosphorylated point mutant (S25E) of the FoxM1 S25 residue, while the mobility was not significantly increased in the cells expressing the other two phosphorylated point mutant S361E and S715E proteins.
  • the above results showed that the phosphorylated point mutant of the S25 residue of FoxM1 promotes the mobility of lung cancer cells and is highly effective.
  • Example 1.8 Evaluation of metastatic and tumorigenic ability of cancer cells by protein containing phosphorylation site and non-phosphorylation point mutation of FoxM1 in animal model
  • the present inventors attempted to evaluate the effect of promoting and suppressing tumorigenesis of cancer cells using cancer cells expressing phosphorylated and non-phosphorylated genes of the FoxM1 S25 residue.
  • A549 lung cancer cells 2 Tumor formation ability was observed.
  • control group Mock; lentivirus treatment group in which target gene is not expressed
  • group administered A549 lung cancer cell line expressing phosphorylated point mutant FoxM1 protein (S25E) non-phosphorylated point group.
  • Cancer cell metastasis and tumor formation were observed in the A549 lung cancer cell line administration group (S25A) expressing mutant FoxM1 protein.
  • the experiment was performed on 5 mice for each experimental group, and the frequency of tumor formation, which is metastatic cancer in the lung, was measured and displayed in a graph (FIG. 5).
  • caspase-3 assay was performed to measure the activity of the caspase-3 enzyme (Figure 5F).
  • Phosphorylated and non-phosphorylated point mutants of FoxM1 A549 cells expressing each protein were treated with 3 ⁇ g/ml doxycycline for 48 hours to induce overexpression of each mutant, and then the cells were lysed to produce the fluorescent substrate (Ac-3) of caspase-3. After reacting for 1 hour with DEVD-AMC), the fluorescence value (Ex. 380nm/Emi.
  • M1 and M2 markers were observed in THP-1 cells.
  • S25E-expressing cancer cells (4x10 4 cells/ml) and THP-1 cells (1.2x10 5 cells/ml) for 48 h, there was no change in the mRNA expression of M1 markers INOS and IL12B in THP-1 cells, but M2 It was observed that the mRNA expression of the markers IL10, CD163, CD206, TGFB1, and VEGFA all increased by more than 2-fold in S25E ( Figure 6A).
  • CD206, TGFB1, and VEGFA are known as markers of M2d-tumor-associated macrophages (M2d-TAM) (JAYASINGAM, Sharmilla Devi, et al., Frontiers in Oncology (2020) 9: 1512.), and FoxM1 S25E is expressed. Since high expression of markers of M2d-tumor-related macrophages was observed in THP-1 cells co-cultured with A549 cells, it was confirmed that THP-1 cells were differentiated into M2d-TAMs due to expression of FoxM1 phosphorylation point mutant.
  • M2d-TAM M2d-tumor-associated macrophages
  • TGFB1 and VEGFA proteins were measured by ELISA in the medium in which A549 cells ( 4x10 cells/ml) expressing the FoxM1 variant were cultured for 48 h, and the results were 5-fold and 4-fold, respectively, in S25E compared to the comparison group (Mock). It was significantly increased, and in S25A, it was expressed at a similar level to the comparison group, so it was observed to be strongly reduced compared to S25E (Figure 6C).
  • an expression suppression system was constructed to suppress FoxM1 expression to observe whether the FoxM1 phosphorylation point mutant (S25E) directly affects differentiation into M2 macrophages.
  • pLKO-puro.1-hFoxM1 plasmid was created using the pLKO-puro.1 vector to create shRNA targeting the nucleotide sequence at positions 709-729 of the human FoxM1 mRNA sequence.
  • the following sequence can be used as a target sequence for producing human FoxM1 shRNA, and an oligonucleotide with the following sequence can be used as a primer for shRNA production.
  • the human FoxM1 mRNA gene accession number in Pubmed is NM_001243088.2 and has 3507 bp.
  • shRNA production the following primers were used: 5'-ccgg-AGCAAGAGATGGAGGAAAAGG-ctcgag-CCTTTT CCTCCATCTCTTGCT-tttttg-3' (forward primer) and 5'-aattcaaaaa-AGCAAG AGATGGAGGAAAAGG-ctcgag-CCTTTTCCTCCATCTCTTGCT-3' (reverse primer).
  • the produced viruses were constructed by infecting A549 cells expressing FoxM1, which are lung cancer cells. After co-culturing lung cancer cells (4x10 4 cells/ml) with suppressed FoxM1 expression and THP-1 cells (1.2x10 5 cells/ml) for 48 h, the expression of mRNA of M2 markers CD163, CD206, and VEGFA was observed. In the S25E cell line in which FoxM1 expression was suppressed, the mRNA expression of M2 markers CD163, CD206, and VEGFA was significantly reduced.
  • A549 cells 4x10 cells/ml: Change the ratio of THP-1 cells to 1:0, 1:2, 1:4, 1:6, and co-culture for 48 h. Then, wash the THP-1 cells and place them on the bottom. The survival rate of attached A549 cells was measured by MTT assay.
  • THP-1 mononuclear cells co-cultured with A549 S25E cells for 48 h.
  • Expression of PD-1 mRNA (CD279), an immune evasion factor, was measured by qRT-PCR ( Figure 7B). It was observed that the mRNA (CD274) expression of the immune evasion factor PD-L1 (CD274) in A549S25E cells expressing the phosphorylation point mutant increased two-fold compared to the control group.
  • A549 S25E A549 S25E cells expressing the FoxM1 S25E phosphorylation point mutant with Jurkat cells, which are T cells
  • Figure 7D The survival rate of A549 S25E cells ( 4x10 cells/ml) expressing the phosphorylated mutant co-cultured with Jurkat cells for 48 h increased by 1.2-fold, 1.3-fold, and 1.3-fold as the ratio of Jurkat cells was increased to 1:0, 1:2, 1:4, and 1:6. While a gradual increase of 1.5-fold was observed, the survival rate of A549 S25A cells was observed to be significantly suppressed compared to A549 cells expressing the original or phosphorylated variant (Figure 7D).
  • A549 S25E cells may also affect the properties of T cells.
  • TILs tumor-infiltrating T lymphocytes
  • Jurkat cells 1.2x10 5 cells
  • A549 S25E cells 4x10 4 cells
  • the expression levels of CD25 and CD29 expressed in TILs were analyzed by qRT-PCR after co-culture for 48 h. As a result, it was observed that the expression of CD25 and CD29 was significantly increased by more than 3-fold in Jurkat cells co-cultured with A549 S25E cells.
  • the present invention provides the effect of suppressing tumor immune evasion response by a non-phosphorylated point mutant of FoxM1.
  • the present inventors used three types of QNAPAETSEE (set #1, set #1, TAT (YGRKKRRQRRR), a type of CPP (Cell-Penetrating Peptide), is used to infiltrate the amino acids of SEQ ID NO: 4), PAETSEEEPK (set #2, SEQ ID NO: 2), and LPVQNAPAET (set #3, SEQ ID NO: 5) into cells. Peptide synthesis was performed ( Figure 8A).
  • the measured fluorescence value was expressed in relative fluorescence units (RFU) as caspase-3 activity to measure the degree of cell death.
  • REU relative fluorescence units
  • RPMI 1640 (10% FBS) containing 3 ⁇ g/ml doxycycline was dispensed, and three types of 5 ⁇ M FoxM1 non-phosphorylated point mutant peptides were simultaneously treated.
  • 500 ⁇ l of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the intensity of staining was measured using the Odyssey infrared imaging system. Assuming that the intensity of staining in the control group was 1, the relative staining intensity in each experimental group was calculated and displayed on a graph.
  • the present inventors sought to evaluate the efficacy of peptides containing three non-phosphorylation point mutations of FoxM1 previously prepared in various cancer types on cancer cell killing.
  • the activity of the caspase-3 enzyme was measured through a caspase-3 assay in the same manner as shown in Figure 8B, and the apoptosis of several cancer cells was observed ( Figure 9B).
  • Cervical cancer cells (HeLa) and liver cancer cells (Hep3B) were treated with three types of 5 ⁇ M peptides each for 48 hours, and each cell treated with the peptides was lysed to produce caspase-3 fluorescent substrate (Ac-DEVD-AMC) and 2 After the time reaction, the fluorescence value (Ex. 380nm/Emi.
  • peptides containing non-phosphorylated point mutations in FoxM1 may have apoptotic properties not only in lung cancer but also in various carcinomas.
  • the present inventors conducted an experiment using FoxM1 non-phosphorylated point mutant peptide #1, which was evaluated as the most effective, to evaluate the inhibitory effect on cancer cell mobility and macrophage differentiation in the metastatic environment caused by a peptide containing a non-phosphorylated point mutation of FoxM1. proceeded. First, to determine whether the FoxM1 non-phosphorylated point mutant peptide penetrates into cells, a FITC fluorescent substance was attached to the C terminus of the peptide to prepare it ( Figure 10A).
  • a cell migration assay was performed to observe the effect of FoxM1 non-phosphorylated point mutant peptide treatment on the mobility of cancer cells in a metastatic environment treated with TGF- ⁇ .
  • 5x10 4 cells of lung cancer cell A549 were dispensed into an 8.0 ⁇ m, 24 well insert, medium containing 10% serum (FBS) was dispensed into the 24 well plate, and the inset was added.
  • FBS 10% serum
  • 0.5 ml of RPMI 1640 (10% FBS) containing 5 ng/ml TGF- ⁇ was used, and 5 ⁇ M non-phosphorylated point mutant peptide was simultaneously treated.
  • 500 ⁇ l of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes.
  • the present inventors investigated changes in the mRNA expression of epithelial-mesenchymal transition markers (EMT markers) by treating lung cancer cells A549, which overexpress FoxM1 phosphorylated point mutant proteins, with FoxM1 non-phosphorylated point mutant peptides. Observation was made through chain reaction (real-time PCR). Cells constructed with an overexpression system for FoxM1 phosphorylated point mutants were treated with 3 ⁇ g/ml doxycycline for 24 hours to induce overexpression, and then treated with FoxM1 non-phosphorylated point mutant peptide for 24 hours to determine the degree of FoxM1 overexpression and epithelial-mesenchymal transition marker (EMT marker). This was confirmed by the mRNA expression level.
  • EMT marker epithelial-mesenchymal transition marker
  • TGF- ⁇ was treated for 48 hours to induce cancer metastasis. Then, using immunoblotting, the protein expression levels of mesenchymal markers N-cadherin, SNAl1, and SNAI2 were significantly increased and epithelial It was confirmed that EMT was activated by seeing that the marker E-cadherin was significantly reduced.
  • EMT was activated by seeing that the marker E-cadherin was significantly reduced.
  • TGF- ⁇ protein binds to the TGF- ⁇ family receptor, it leads to phosphorylation of the receptor and activation of the SMAD complex, thereby activating the EMT program (Valcourt Ulrich, et al., Mol Biol Cell (2005) 16(4) 1987 -2002). In this system as well, it was observed that the expression of phosphorylated Smad-2 increased upon treatment with TGF- ⁇ . Under these conditions, we observed that the protein expression of FoxM1 also increased (Figure 11).
  • lung cancer cells were cultured as follows to infect lung cancer cells with a lentivirus expressing the FoxM1 circular protein.
  • the cells were first infected with pLVX-Tet3G-expressing lentivirus and treated with 500 ⁇ g/ml of G418 for 5 days to select infected cells.
  • the selected A549 Tet3G cells were infected with lentiviruses expressing the original form (WT) and control group (Mock) of FoxM1, and then treated with 2 ⁇ g/ml puromycin for 48 hours to construct a stabilized cell line.
  • the constructed cells were treated with 2 ⁇ g/ml doxycycline to induce the original expression of FoxM1, and protein expression was observed using immunoblot to determine whether the original FoxM1 was expressed.
  • a migration assay was performed to observe the effect on cancer cell mobility in lung cancer cells A549 expressing the FoxM1 circular protein.
  • 5 I added the back inset.
  • 0.5 ml of RPMI 1640 (10% FBS) containing 5 ng/ml TGF- ⁇ was used for the positive control.
  • 500 ⁇ l of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the intensity of staining was measured using the Odyssey infrared imaging system. Assuming that the staining intensity (intensity) of the control group was 1, the relative staining intensity of the positive group and the experimental group was calculated and displayed on a graph.
  • Matrigel was completely dissolved at 4°C for 16 to 20 hours, and then Matrigel was diluted to 1 mg/ml with cold serum-free RPMI 1640 (4°C). 100 ⁇ l of Matrigel mixture (1 mg/ml) was added to an 8.0 mm 24 well insert and hardened in a 37°C incubator for 12-20 hours. Lung cancer cells A549 expressing the control (Mock) and experimental group FoxM1 prototype (WT) protein were diluted in serum free RPMI 1640 (36°C) at a cell count of 1X10 cells/well and dispensed onto the hardened Matrigel insert. Here, 0.5 ml/well of warm RPMI 1640 (10% FBS) at 36°C was dispensed.
  • 0.5 ml of 36°C RPMI 1640 (10% FBS) containing 5 ng/ml TGF- ⁇ was used. After that, the medium was changed once every three days and the degree of invasion was observed. On the 7th day, when it was observed that cancer cell invasion had sufficiently occurred, the medium was removed, washed with 1XPBS, and the cells inside the insert were scraped off with a cotton swab. It was washed with 1XPBS to remove any remaining cells and matrigel inside the insert.
  • CD206, TGFB1, and VEGFA are known as markers of M2d-tumor-associated macrophages (M2d-TAM) (JAYASINGAM, Sharmilla Devi, et al., Frontiers in Oncology (2020) 9: 1512.), and FoxM1 circular protein is expressed.
  • M2d-TAM M2d-tumor-associated macrophages
  • JYASINGAM Javamilla Devi, et al., Frontiers in Oncology (2020) 9: 1512.
  • FoxM1 circular protein is expressed.
  • High expression of markers of M2d-tumor-related macrophages was observed in THP-1 cells co-cultured with A549 cells, indicating that THP-1 cells were differentiated into M2d-TAMs due to the expression of the original FoxM1.
  • shRNA and a lentivirus containing it were produced.
  • primers were prepared as follows. Use 5'- ccgg- CAT CAG AGG AGG AAC CTA AGA - ctcgag - TCT TAG GTT CCT CCT CTG ATG - tttttg - 3' as a forward primer targeting the nucleotide sequence at positions 187-207 (target #1) of the FoxM1 mRNA sequence.
  • the forward primer targeting the nucleotide sequence at position 709-729 (target #2) of the FoxM1 mRNA sequence is 5' - ccgg - AGC AAG AGA TGG AGG AAA AGG- ctcgag - CCT TTT CCT CCA TCT CTT GCT - tttttg - 3' was used, and 5' - aattcaaaaa - AGC AAG AGA TGG AGG AAA AGG - ctcgag - CCT TTT CCT CCA TCT CTT GCT - 3' was used as the reverse primer.
  • pLKO-puro.1-shFoxM1 plasmid was created using the pLKO-puro.1 vector. This was expressed through HEK293 cell transfection along with pHR'-CMV-VSVG and pHR'-CMV-deltaR8.2, and the culture medium of the cells was collected to produce lentivirus. The lentivirus was concentrated using a centrifuge. To confirm virus expression, A549 cells were cultured at 5 After 24 hours of infection, shFoxM1-infected cells were selected by treating them with 2 ⁇ g/ml puromycin for 48 hours.
  • the present inventor confirmed the mRNA and protein expression levels of FoxM1 in cells infected with each selected FoxM1 shRNA by treatment with puromycine 2 ⁇ g/ml for 48 hours. .
  • the expression of FoxM1 mRNA was decreased in lung cancer cells A549 infected with each FoxM1 shRNA (shFoxM1) compared to cells infected with control shRNA (shCtrl), which showed that FoxM1 expression was suppressed.
  • shRNA targeting the nucleotide sequence at positions 709-729 (target #2) in the FoxM1 mRNA sequence was observed to have the best effect in suppressing FoxM1 expression.
  • the present inventor used shRNA targeting the nucleotide sequence at position 709-729 (target #2) in the FoxM1 mRNA sequence when studying the inhibition of FoxM1 expression below.
  • the present inventors conducted a cell migration assay to determine whether inhibition of FoxM1 expression by FoxM1 shRNA treatment inhibits lung cancer cell mobility.
  • lung cancer cells A549 were infected with control shRNA (shCtrl) and FoxM1 shRNA (shFoxM1) viruses, respectively.
  • shCtrl control shRNA
  • shFoxM1 shRNA shFoxM1 shRNA
  • Relative moving distance (%) measured value in the experimental group x 100 / measured value in the control group
  • the present inventors observed that cancer metastasis induced by treatment with TGF- ⁇ suppressed FoxM1 mRNA expression by FoxM1 shRNA treatment and changed the mRNA and protein expressions of epithelial-mesenchymal transition markers and related factors.
  • caspase-3 assay was performed to measure the activity of caspase-3 enzyme (Figure 14F).
  • A549 cells were infected with viruses expressing shCtrl, shFoxM1#187, and shFoxM1#709 for 48 hours, followed by puromycin selection for 48 hours, and cells were obtained and lysed.
  • the fluorescent substrate Ac-DEVD-AMC
  • the fluorescence value was measured using an electron spectrometer (spectramax M4 system).
  • the measured fluorescence value is expressed as relative fluorescence units (RFU) for caspase-3 activity, indicating the degree of cell death.
  • REU relative fluorescence units
  • the present inventors observed whether inhibition of FoxM1 expression by FoxM1 shRNA treatment resulted in a decrease in FoxM1 expression in phosphorylation point mutants of FoxM1.
  • lung cancer cells A549 expressing the FoxM1 phosphorylation point mutation were infected with control shRNA (shCtrl) and FoxM1 shRNA (shFoxM1) viruses, respectively.
  • shCtrl control shRNA
  • shFoxM1 shRNA FoxM1 shRNA
  • lung cancer cell A549 expressing the FoxM1 phosphorylation point mutation (S25E) was infected with control shRNA (shCtrl) and FoxM1 shRNA (shFoxM1) viruses, respectively, through real-time polymerase chain reaction (Real-time PCR) of IFITM1.
  • control shRNA shCtrl
  • FoxM1 shRNA shFoxM1 shRNA
  • Real-time PCR real-time polymerase chain reaction
  • Example 2.9 Evaluation of the effect of suppressing epithelial-mesenchymal transition and differentiation of macrophages in cells overexpressing phosphorylation point mutations of FoxM1 by treatment with Thiostrepton, a FoxM1 inhibitor
  • the present inventors observed whether inhibition of FoxM1 expression by treatment with Thiostrepton, a FoxM1 inhibitor, inhibits epithelial-mesenchymal transition and differentiation ability of macrophages in cells overexpressing a phosphorylated point mutant protein of FoxM1.
  • lung cancer cell A549 control (Mock) and cells expressing FoxM1 phosphorylation point mutation (S25E) were treated with Thiostrepton, a FoxM1 inhibitor, at a concentration of 5 ⁇ M for 48 h, and epithelial-mesenchymal growth was achieved through real-time polymerase chain reaction (Real-time PCR).
  • Real-time PCR real-time polymerase chain reaction
  • Expression of migration-related mesenchymal markers CDH2, vimentin, SNAI1, and SNAI2 was observed to be significantly reduced in the group treated with Thiostrepton, a FoxM1 inhibitor (Figure 16A).

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Abstract

The present invention relates to use of a substance containing a point-mutation FoxM1 protein and peptide for inhibiting the growth, mobility, and invasiveness of cancer cells. In addition, the present invention relates to a use of the substance containing a point-mutation FoxM1 protein and peptide for inhibiting malignancy in a tumor microenvironment by suppressing differentiation into tumor-associated macrophages in the tumor microenvironment. The present invention relates to use as an apoptosis-inducing agent using the action of sensitively inhibiting apoptosis of cancer cells in metastatic cancer cells as well as common solid cancers. The present invention can be effectively used to treat various solid cancers and metastatic cancers.

Description

FOXM1 돌연변이체 또는 FOXM1 SHRNA를 포함하는 암 치료용 약학 조성물 Pharmaceutical composition for treating cancer comprising FOXM1 mutant or FOXM1 SHRNA
본 발명은 FoxM1의 점 돌연변이 또는 FoxM1 점 돌연변이를 포함하는 펩티드, 또는 FoxM1에 특이적으로 결합하는 shRNA를 이용한 암세포 성장과 침습성 및 전이성을 억제하기 위한 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for inhibiting cancer cell growth, invasiveness, and metastasis using a point mutation of FoxM1, a peptide containing a FoxM1 point mutation, or shRNA that specifically binds to FoxM1.
암은 암의 진행단계에 따라 나눌 수 있으며, 특히 단계에 따라 암의 전이성은 치료방법을 결정하는데 중요한 기준이 된다. 암의 크기도 중요하지만, 1차성 암인 원발소 암과 주변 림프절 또는 장기 등으로 이동하는 전이성 암으로 나누어 그 치료전략을 검토해야 한다. 잘 알려진 바와 같이 원발소 암을 치료하였다 하더라도, 전이를 막을 수 없다면 생존율은 매우 낮아진다. 전이성 암은 암종에 따라 차이가 있으나, 암환자 사망의 90%까지 차지할 수 있어 그 예후가 매우 좋지 않다 (Dillekas, et al., Cancer Med 8 (2019) 5574-5576; Khan, I. and Steeg, P.S., Lab Invest 98 (2018) 198-210). 암의 성장이 지속될수록 주변 림프절 및 조직에 암 조직에서 악성화 및 전이를 돕는 사이토카인의 분비를 촉진하고 이는 결과적으로 암 전이를 증가시키는 원인으로 작용하게 된다. 암을 제거하는 기능을 지닌 대식세포도 암세포가 분비하는 IL6, VEGFA, TGFB1 등으로 종양관련 대식세포 (Tumor-associated macrophage; TAM)로 분화되어 오히려 암 생존을 돕는 세포로 작용하는 것으로 알려져 있다 (Murray P.J. Annu Rev. Physiol. (2017) 79:541-566; Jeon SH et al., J. Leukoc Biol. (2007) 81(2) 557-566; Wheeler KC et al., PLoS One (2018) 13(1):e0191040). 종양의 크기가 클수록 전이 비율이 높은 것으로 알려져 있으나, 종양의 크기가 작음에도 전이가 되는 경우가 있어서, 암의 증식과 전이간의 관계는 아직까지 명확하게 규명되어 있지 않다 (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Shibue, T. and Weinberg, Semin Cancer Biol 21 (2011) 99-106). 항암치료에 있어 암세포의 증식을 억제하는 것을 기본으로 하고 있으나 전이를 막지 못할 경우 그 예후가 현저히 낮아진다는 점을 고려한다면, 전이암의 치료를 위한 암세포의 전이 및 침윤을 효과적으로 억제하는 항암 치료제의 개발이 필요하다. Cancer can be divided according to the stage of cancer progression, and in particular, the metastasis of cancer depending on the stage is an important criterion in determining treatment methods. Although the size of the cancer is important, treatment strategies should be reviewed by dividing it into primary cancer, which is the primary cancer, and metastatic cancer that moves to nearby lymph nodes or organs. As is well known, even if the primary cancer is treated, the survival rate is very low if metastasis cannot be prevented. Metastatic cancer varies depending on the type of cancer, but can account for up to 90% of deaths in cancer patients, so the prognosis is very poor (Dillekas, et al., Cancer Med 8 (2019) 5574-5576; Khan, I. and Steeg, P.S., Lab Invest 98 (2018) 198-210). As the cancer continues to grow, it promotes the secretion of cytokines that help malignancy and metastasis in the surrounding lymph nodes and tissues, which ultimately acts as a cause of increased cancer metastasis. Macrophages, which have the function of eliminating cancer, are also known to differentiate into tumor-associated macrophages (TAM) due to IL6, VEGFA, and TGFB1 secreted by cancer cells, and act as cells that help cancer survive (Murray) P. J. Physiol (2017) 79:541-566; J. Leukoc Biol (2007) 81 (2) 557-566; PLoS One (2018) 1):e0191040). It is known that the larger the tumor, the higher the rate of metastasis. However, in some cases, metastasis occurs even when the tumor size is small, and the relationship between cancer proliferation and metastasis has not yet been clearly identified (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Shibue, T. and Weinberg, Semin Cancer Biol 21 (2011) 99-106). Anticancer treatment is based on suppressing the proliferation of cancer cells, but considering that the prognosis is significantly lower if metastasis is not prevented, development of an anticancer treatment that effectively suppresses metastasis and invasion of cancer cells for the treatment of metastatic cancer is necessary. This is needed.
FoxM1은 Forkhead box (Fox) 전사 인자의 큰 계열에 속하며 'wingled-helix' 로 불리는 DNA 결합 도메인이 공통적으로 존재하는 전사조절인자이다(Kaufmann, E. and Knoechel, W., Mech Dev 57 (1996) 3-20). FoxM1 전사 인자는 세포 증식, 세포 주기 진행, 세포 분화, DNA 손상 복구, 조직 항상성, 혈관 신생 및 세포 사멸을 포함한 광범위한 생물학적 과정의 조절에 필수적인 역할을 한다. FoxM1은 세포주기 중 S기 및 G2/M기에서 그 발현이 정점을 이루며, 세포주기 진행에서 핵심적인 역할을 하는 것으로 알려져 있다(Laoukili, J. et al., Nat Cell Biol 7 (2005) 126-136). 이는 PLK1, cyclin B1, Nek2 및 CENPF와 같은 많은 G2/M 특이적 유전자의 발현을 조절한다(WANG, I.-Ching et al., Mol Cell Biol (2005) 25.24:10875-10894). 또한 유사분열 전사 인자 FoxM1은 SNAI1 및 SNAI2를 포함하는 EMT 전사 인자의 활성화에 의해 추정되는 EMT 조절자이다. 상피에서 중간엽 유사 전이, 세포 이동 및 전이성 틈새 형성을 포함하여 종양 전이의 다양한 단계에 관여하는 유전자의 발현을 자극할 수 있다. FoxM1의 증가된 발현은 간암(YU, Chun-Peng et al. Molecular medicine reports 16.4 (2017) 5181-5188), 전립선암(Kalin, Tanya V. et al., Cancer research 66.3 (2006) 1712-1720), 대장암(Yoshida, Yuichi et al., Gastroenterology 132.4 (2007) 1420-1431), 뇌암(Liu, Mingguang et al., Cancer research 66.7 (2006) 3593-3602), 유방암(Millour, Julie and E. W. Lam. Breast Cancer Research 12.1 (2010) 1-1), 폐암(Wang, I-Ching et al. PLoS One 4.8 (2009): e6609), 결장암, 췌장암, 피부암, 자궁경부암, 난소암, 구강암, 혈액암 및 신경계를 비롯한 다양한 악성 종양에서도 관찰된다(BARGER, Carter J. et al., Cancers (2019) 11.2: 251). 암의 게놈 전체 유전자 발현 프로파일링은 FoxM1을 인간 고형 종양에서 가장 일반적으로 상향 조절되는 유전자 중 하나로 독립적이고 일관되게 확인했다. 이러한 발견은 종양 형성에서 FoxM1의 주요 역할을 함을 나타낸다.FoxM1 belongs to the large family of forkhead box (Fox) transcription factors and is a transcriptional regulator with a common DNA binding domain called 'wingled-helix' (Kaufmann, E. and Knoechel, W., Mech Dev 57 (1996) 3-20). The FoxM1 transcription factor plays an essential role in the regulation of a wide range of biological processes, including cell proliferation, cell cycle progression, cell differentiation, DNA damage repair, tissue homeostasis, angiogenesis, and apoptosis. FoxM1's expression peaks in the S phase and G2/M phase of the cell cycle, and is known to play a key role in cell cycle progression (Laoukili, J. et al., Nat Cell Biol 7 (2005) 126- 136). It regulates the expression of many G2/M-specific genes such as PLK1, cyclin B1, Nek2 and CENPF (WANG, I.-Ching et al., Mol Cell Biol (2005) 25.24:10875-10894). Additionally, the mitotic transcription factor FoxM1 is a putative EMT regulator by activation of EMT transcription factors including SNAI1 and SNAI2. It can stimulate the expression of genes involved in various stages of tumor metastasis, including epithelial to mesenchymal-like transition, cell migration, and metastatic niche formation. Increased expression of FoxM1 is associated with liver cancer (YU, Chun-Peng et al. Molecular medicine reports 16.4 (2017) 5181-5188) and prostate cancer (Kalin, Tanya V. et al., Cancer research 66.3 (2006) 1712-1720). , colon cancer (Yoshida, Yuichi et al., Gastroenterology 132.4 (2007) 1420-1431), brain cancer (Liu, Mingguang et al., Cancer research 66.7 (2006) 3593-3602), breast cancer (Millour, Julie and E. W. Lam. Breast Cancer Research 12.1 (2010) 1-1), lung cancer (Wang, I-Ching et al. PLoS One 4.8 (2009): e6609), colon cancer, pancreatic cancer, skin cancer, cervical cancer, ovarian cancer, oral cancer, blood cancer, and nervous system. It is also observed in various malignant tumors, including (BARGER, Carter J. et al., Cancers (2019) 11.2: 251). Genome-wide gene expression profiling of cancer has independently and consistently identified FoxM1 as one of the most commonly upregulated genes in human solid tumors. These findings indicate a key role for FoxM1 in tumorigenesis.
FoxM1은 기능적으로는 성장 및 분열하는 세포에서 발현이 증가되는데 특히 세포주기 중 세포분열기에서의 발현과 활성이 정점을 이루고 있다. 따라서 빠르게 성장하는 암세포에서 또한 그 발현율이 높은 것으로 알려져 있다 (Liao, Guo-Bin et al., Cell Communication and Signaling 16.1 (2018): 1-15). 최근 다양한 암종에서 그 단계에 따라 발현이 증가되는 것이 보고되면서 FoxM1의 성장을 주도하는 기능 외에 암전이에서도 그 관련성이 제시되고 있다. 특히 대장암(Fei, BaoYing et al., Oncology Letters 14.6 (2017): 6553-6561), 폐암(Wei, Ping et al., Int J Biol Sci 11.2 (2015): 186), 난소암(Chan, David W. et al., Oncogene 36.10 (2017): 1404-1416) 등에서 stage가 높을수록 그 발현이 증가되어 있음이 보고되었다(Li, Lijun et al., Oncotarget 8.19 (2017): 32298). 따라서 stage가 높은 암에서 발현이 높은 FoxM1의 발현을 억제하게 되면, 원발성 암의 성장 및 전이암으로의 전환을 억제할 수 효과가 뛰어날 것으로 기대된다. Functionally, the expression of FoxM1 increases in growing and dividing cells, and its expression and activity peak in the cell division phase of the cell cycle. Therefore, it is known to have a high expression rate in rapidly growing cancer cells (Liao, Guo-Bin et al., Cell Communication and Signaling 16.1 (2018): 1-15). Recently, it has been reported that the expression of FoxM1 increases depending on the stage in various carcinomas, suggesting that FoxM1 is related to cancer metastasis in addition to its function of leading growth. In particular, colon cancer (Fei, BaoYing et al., Oncology Letters 14.6 (2017): 6553-6561), lung cancer (Wei, Ping et al., Int J Biol Sci 11.2 (2015): 186), and ovarian cancer (Chan, David W. et al., Oncogene 36.10 (2017): 1404-1416) reported that the expression increased as the stage increased (Li, Lijun et al., Oncotarget 8.19 (2017): 32298). Therefore, suppressing the expression of FoxM1, which is highly expressed in high-stage cancer, is expected to be highly effective in suppressing the growth of primary cancer and conversion to metastatic cancer.
전이암은 암종에 따라서는 암환자 사망의 90%를 차지하고 있어 환자의 생존율에 대한 그 위험도가 익히 잘 알려져 있다 (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Khan, I. and Steeg, P.S., Lab Invest 98 (2018) 198-210). 암전이는 원발소 암이 생존에 적합한 환경으로의 이동 결과로 나타나는 현상으로, 전이를 막을 수 없다면 원발소 암의 제거만으로는 암의 재발 등에 의해서 환자의 생존율이 매우 낮아진다. 암의 크기가 커질수록 주변 림프절과 장기로의 전이 비율이 높아지는 것으로 알려져 있으나, 암의 크기가 작음에도 불구하고 전이가 되는 경우가 있어, 아직까지 암의 전이와 증식의 관계는 여전히 명확하게 규명되어 있지 않다 (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Shibue, T. and Weinberg, Semin Cancer Biol 21 (2011) 99-106).Metastatic cancer accounts for 90% of deaths in cancer patients depending on the type of cancer, so its risk to patient survival is well known (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Khan, I and Steeg, P.S., Lab Invest 98 (2018) 198-210). Cancer metastasis is a phenomenon that occurs as a result of the primary cancer moving into an environment suitable for survival. If metastasis cannot be prevented, the patient's survival rate is very low due to cancer recurrence, etc., simply by removing the primary cancer. It is known that the rate of metastasis to nearby lymph nodes and organs increases as the size of the cancer increases. However, in some cases, the cancer metastasizes even though the size is small, and the relationship between cancer metastasis and proliferation is still not clearly identified. There is no (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Shibue, T. and Weinberg, Semin Cancer Biol 21 (2011) 99-106).
Polo-like kinase 1(PLK1)은 세포성장을 조절하는 대표적인 세포분열 인자로 다양한 고형암과 혈액암에서 그 발현이 높아, 다양한 암의 진단에 활용이 되고 있다. 최근 발암뿐만 아니라 전이를 유발할 수 있다는 연구결과가 보고되면서 (Shin et al., Oncogene (2020) 39(4) 767-785; Wu et al., Elife (2016) doi: 10.7554/eLife.10734.) 전이의 타겟분자로서 연구되고 있다. 이러한 PLK1 특성을 기반으로 PLK1에 대한 억제제 개발을 통한 항암제 개발 연구가 다국적기업을 중심으로 경쟁적으로 진행되고 있다(Yim, Anti-Cancer Drugs 24(2013) 999-1006; Zhang, J. Med. Chem. 65 (2022) 10133-10160). 구조적으로 PLK1은 ATP가 결합할 수 있는 ATP-결합 도메인을 지닌 인산화효소 활성 도메인과 기질과 결합하는 폴로박스 도메인을 지니고 있다. 인산화효소 활성 도메인에 위치한 Thr 210 잔기에서 인산화가 일어나면 PLK1의 활성화가 유도되며 이는 폴로박스 도메인에 결합한 기질 단백질의 Ser/Thr 잔기를 인산화하는 효소이다(Barr et al., Nat Rev Mol Cell Biol 5 (2004) 429-440). PLK1은 기능적으로 세포의 성장 및 분열 과정에서 그 발현이 증가되는데 특히 세포 분열기에서 가장 높으며 그 활성이 정점을 이룬다. 따라서 빠르게 성장하는 암세포에서 또한 그 발현율이 높은 것으로 알려져 있다(Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31-39; Barr et al., Nat Rev Mol Cell Biol 5 (2004) 429-440). 최근의 연구에서, 다양한 암종의 단계별 악성화 과정에서, 대부분 그 발현이 단계가 증가함에 따라 증가되는 것이 임상 결과들을 바탕으로 보고되었다. 특히 전립선암, 비소세포폐암, 자궁내막암, 대장암, 난소암, 후두암 등에서 악성 단계가 높을수록 그 발현이 증가되어 있음이 보고되었다(Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31-39; Kim et al., Exp Mol Med, 54 (2022) 414-425). 최근의 연구에 따르면 활성형의 PLK1 만으로도 암 전이성을 증가시키는 데 관여하고 있음이 보고되었고 (Shin et al., Oncogene (2020) 39(4) 767-785; Cai et al., Am J Transl Res 8 (2016) 4172-4183; Wu et al., Elife (2016) doi: 10.7554/eLife.10734.), 이를 기반으로 PLK1 타겟팅이 전이성 암 치료를 위한 치료전략으로 부각되고 있다. 따라서, 본 연구자들은 원발소 암뿐만 아니라 전이성 암 치료에 PLK1이 직접적으로 인산화시키는 기질의 기능 조절을 통하여 항암제 개발 전략을 구축하였다. Polo-like kinase 1 (PLK1) is a representative cell division factor that regulates cell growth, and its expression is high in various solid and blood cancers, so it is used in the diagnosis of various cancers. Recently, research results have reported that it can cause not only carcinogenesis but also metastasis (Shin et al., Oncogene (2020) 39(4) 767-785; Wu et al., Elife (2016) doi: 10.7554/eLife.10734.) It is being studied as a target molecule for metastasis. Based on these PLK1 characteristics, anticancer drug development research through the development of inhibitors for PLK1 is being competitively conducted by multinational companies (Yim, Anti-Cancer Drugs 24 (2013) 999-1006; Zhang, J. Med. Chem. 65 (2022) 10133-10160). Structurally, PLK1 has a kinase activity domain with an ATP-binding domain that can bind ATP and a polo box domain that binds substrates. When phosphorylation occurs at the Thr 210 residue located in the phosphatase activity domain, activation of PLK1 is induced, which is an enzyme that phosphorylates Ser/Thr residues of substrate proteins bound to the Polobox domain (Barr et al., Nat Rev Mol Cell Biol 5 ( 2004) 429-440). Functionally, the expression of PLK1 increases during cell growth and division, and its activity is highest and peaks during cell division. Therefore, it is known to have a high expression rate in rapidly growing cancer cells (Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31-39; Barr et al., Nat Rev Mol Cell Biol 5 (2004) 429-440 ). In recent studies, it has been reported based on clinical results that in the stage-by-stage malignant transformation of various carcinomas, the expression increases as the stage increases. In particular, it has been reported that the expression increases as the malignant stage increases in prostate cancer, non-small cell lung cancer, endometrial cancer, colon cancer, ovarian cancer, and laryngeal cancer (Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31 -39; Kim et al., Exp Mol Med, 54 (2022) 414-425). Recent studies have reported that the active form of PLK1 alone is involved in increasing cancer metastasis (Shin et al., Oncogene (2020) 39(4) 767-785; Cai et al., Am J Transl Res 8 (2016) 4172-4183; Wu et al., Elife (2016) doi: 10.7554/eLife.10734.), based on this, targeting PLK1 is emerging as a therapeutic strategy for treating metastatic cancer. Therefore, the present researchers established an anticancer drug development strategy for the treatment of not only primary cancer but also metastatic cancer by regulating the function of substrates directly phosphorylated by PLK1.
암전이 과정은 다단계로 초기 침습성과 이동성은 암세포의 EMT (Epithelial-mesenchymal transition)과정을 거쳐 획득되는 것으로 알려져 있다 (Dongre, Anushka, and Robert A. Weinberg. Nature reviews Molecular cell biology 20.2 (2019): 69-84). EMT 과정은 상피성의 세포가 세포간의 긴밀한 결합과 같은 특성을 잃고 이동능과 침습성을 가지는 중간엽 세포로 전환되는 과정이다. 이 때 세포내 인자들의 변화를 보면 epithelial marker인 E-cadherin이 줄어들면서 mesenchymal marker 인 N-cadherin, vimentin, SNAI1, SNAI2 등의 증가가 관찰된다. 암의 치료에 있어, 암 세포의 증식 억제와 전이의 억제가 항상 같이 나타나는 효과가 아니고, 암의 전이를 억제할 수 있다면 많은 암의 치료 효율을 비약적으로 개선할 수 있다는 측면에서, 전이성 암의 치료를 위한 암의 전이 및 침윤을 효과적으로 억제하는 치료 타겟 또는 치료제의 개발이 필요하다.The cancer metastasis process is multi-step, and it is known that initial invasiveness and mobility are acquired through the EMT (Epithelial-mesenchymal transition) process of cancer cells (Dongre, Anushka, and Robert A. Weinberg. Nature reviews Molecular cell biology 20.2 (2019): 69 -84). The EMT process is a process in which epithelial cells lose characteristics such as tight intercellular bonds and are converted into mesenchymal cells with migratory and invasive properties. At this time, looking at the changes in intracellular factors, the epithelial marker E-cadherin decreases while the mesenchymal markers N-cadherin, vimentin, SNAI1, and SNAI2 increase. In the treatment of cancer, inhibition of cancer cell proliferation and inhibition of metastasis do not always have the same effect, and if cancer metastasis can be inhibited, the treatment efficiency of many cancers can be dramatically improved. There is a need to develop treatment targets or treatments that effectively inhibit cancer metastasis and invasion.
본 발명자들은 PLK1에 의한 FoxM1의 인산화부위 변이체를 개발하여 이를 기반으로 하는 유전자 및 펩티드 치료 기반의 항암제 개발에 노력한 결과, FoxM1의 PLK1 인산화 부위인 Ser25의 비인산화 점 돌연변이체가 폐암 세포의 전이성 및 침습성에 대한 억제 효과가 있음을 관찰하였다. 또한 FoxM1의 Ser25번의 인산화 점 돌연변이체가 종양미세환경에 존재하는 단핵구를 종양주변으로 이동시켜 종양의 생존을 도와주는 종양 관련 대식세포 (Tumor-associated macrophage)로의 분화를 촉진시키며 혈관신생의 주요인자인 VEGFA의 발현을 증가시키고, 면역회피를 통한 T 세포의 암세포 면역회피를 돕는 일련의 종양미세환경에서의 악성화를 유발시키는 것을 확인한 바, 이를 역으로 이용하여 FoxM1의 비인산화 점 돌연변이체 단백질 및 펩티드를 개발하여 각종 고형암의 전이성, 침습성 및 종양 형성을 차단하고, 종양미세환경에서의 종양의 면역회피를 돕는 면역세포들의 활성을 감소시켜 전이성 암치료에 유용하게 사용될 수 있음을 확인함으로써 본 발명을 완성하였다.The present inventors developed a variant of the phosphorylation site of FoxM1 by PLK1 and made efforts to develop anticancer drugs based on gene and peptide therapy based on it. As a result, the non-phosphorylated point mutant of Ser25, the PLK1 phosphorylation site of FoxM1, was found to be effective in the metastatic and invasive properties of lung cancer cells. It was observed that there was an inhibitory effect on In addition, the phosphorylated point mutant at Ser25 of FoxM1 moves monocytes present in the tumor microenvironment to the periphery of the tumor, promoting differentiation into tumor-associated macrophages that help the tumor survive, and VEGFA, a key factor in angiogenesis, promotes differentiation. It was confirmed that it increases the expression of and induces malignancy in a series of tumor microenvironments that help T cells evade cancer cells through immune evasion. Using this in reverse, non-phosphorylated point mutant proteins and peptides of FoxM1 were developed. The present invention was completed by confirming that it can be effectively used in the treatment of metastatic cancer by blocking the metastasis, invasiveness, and tumor formation of various solid cancers and reducing the activity of immune cells that help the tumor's immune evasion in the tumor microenvironment.
또한 본 발명자들은 종양의 성장과 전이 조절뿐만 아니라 종양미세환경에서 종양관련 대식세포로의 분화를 촉진시키는 FoxM1의 작용을 차단하기 위하여, FoxM1의발현 억제성의 FoxM1 shRNA와 FoxM1 작용 억제제 thiostepton이 폐암의 전이성, 침습성, 종양 형성, 종양관련 대식세포의 분화를 현저히 감소시키는 결과를 발견하여, FoxM1 shRNA와 FoxM1 억제제 thiostepton가 각종 고형암의 전이성, 침습성 및 종양 형성을 차단하고 감소시켜 전이성 암치료에 유용하게 사용될 수 있음을 확인함으로써 본 발명을 완성하였다.In addition, in order to block the action of FoxM1, which not only regulates tumor growth and metastasis but also promotes differentiation into tumor-related macrophages in the tumor microenvironment, the present inventors used FoxM1 shRNA, which suppresses FoxM1 expression, and thiostepton, a FoxM1 inhibitor, to inhibit the metastatic properties of lung cancer. , found that it significantly reduced the invasiveness, tumor formation, and differentiation of tumor-related macrophages, and found that FoxM1 shRNA and the FoxM1 inhibitor thiostepton can be useful in the treatment of metastatic cancer by blocking and reducing the metastasis, invasiveness, and tumor formation of various solid tumors. The present invention was completed by confirming that it exists.
본 발명은 점 돌연변이를 포함하는 FoxM1 단백질 및 펩티드, 또는 FOXM1에 특이적으로 결합하여 FOXM1의 발현을 억제시킬 수 있는 shRNA가 고형암에서 성장도와 침습성과 전이성을 억제하며, 비인산화 점 돌연변이를 포함하는 FoxM1 단백질, 또는 이의 단편, 또는 FOXM1에 특이적으로 결합하는 shRNA에 의해 폐암 세포에서 나타나는 암세포의 전이성, 침습성 및 종양미세환경에서의 종양의 면역회피를 돕는 면역세포들의 활성에 대한 강력한 억제 작용을 이용한 암 치료제를 제공하기 위한 것이다. The present invention provides that FoxM1 protein and peptide containing a point mutation, or shRNA that can specifically bind to FOXM1 and inhibit the expression of FOXM1, suppresses growth, invasiveness, and metastasis in solid tumors, and FoxM1 containing a non-phosphorylated point mutation Cancer using a strong inhibitory effect on the activity of immune cells that help the metastasis and invasiveness of cancer cells in lung cancer cells and immune evasion of tumors in the tumor microenvironment by proteins, fragments thereof, or shRNA that specifically binds to FOXM1. It is intended to provide a cure.
본 발명은 서열번호 1의 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된 폴리펩티드를 포함하는, 암 치료용 약학 조성물을 제공한다. 본 발명의 일 실시예에서, 상기 비인산화 아미노산은 Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His, 또는 Lys이고, 본 발명의 다른 실시예에서, 상기 암은 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 또는 뇌하수체 선종이며, 본 발명의 또 다른 실시예에서, 상기 약학 조성물은 암세포의 성장성, 이동성, 침습성, 및 전이성으로 구성된 군으로부터 선택된 하나 이상을 억제한다. The present invention provides a pharmaceutical composition for treating cancer, comprising a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with a non-phosphorylated amino acid. In one embodiment of the present invention, the non-phosphorylated amino acid is Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His, or Lys, and in another embodiment of the present invention In examples, the cancer includes bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, and ovarian cancer. , rectal cancer, cancer of the anal area, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of sexual and reproductive organs, Hodgkin's disease , esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal tumor, nerve glioma, meningioma, or pituitary adenoma, and in another embodiment of the present invention, the pharmaceutical composition inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
본 발명의 일실시예에서는 서열번호 1의 24번째 아미노산 Pro 내지 27번째 아미노산 Thr을 포함하고, 연속된 10개 이상의 아미노산을 포함하며, 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된, 폴리펩티드를 포함하는 폴리펩티드를 제공하고, 본 발명의 다른 실시예에서, 상기 폴리펩티드는 서열번호 2, 4, 또는 5로 표시되는 아미노산 서열을 포함한다. In one embodiment of the present invention, a polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, 10 or more consecutive amino acids, and the 25th amino acid Ser is substituted with a non-phosphorylated amino acid. In another embodiment of the present invention, a polypeptide is provided, wherein the polypeptide comprises an amino acid sequence represented by SEQ ID NO: 2, 4, or 5.
본 발명의 일 실시예에서는 서열번호 1의 24번째 아미노산 Pro 내지 27번째 아미노산 Thr을 포함하고, 연속된 10개 이상의 아미노산을 포함하며, 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된 폴리펩티드를 포함하는, 암 치료용 약학 조성물을 제공한다. 본 발명의 일 실시예에서, 상기 폴리펩티드는 서열번호 2, 4, 또는 5로 표시되는 아미노산 서열을 포함하고, 본 발명의 다른 실시예에서, 상기 암은 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 또는 뇌하수체 선종이고, 본 발명의 다른 실시예에서, 상기 약학 조성물은 암세포의 성장성, 이동성, 침습성, 및 전이성으로 구성된 군으로부터 선택된 하나 이상을 억제한다. In one embodiment of the present invention, a polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, 10 or more consecutive amino acids, and the 25th amino acid Ser is substituted with a non-phosphorylated amino acid, A pharmaceutical composition for treating cancer is provided. In one embodiment of the present invention, the polypeptide includes an amino acid sequence represented by SEQ ID NO: 2, 4, or 5, and in another embodiment of the present invention, the cancer is bone cancer, blood cancer, lung cancer, small cell lung cancer, arsenic cancer, etc. Cellular lung cancer, squamous cell carcinoma, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, and prostate cancer. , uterine cancer, endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer. , soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasm of the central nervous system (CNS), neuroectodermal cancer, spinal tumor, glioma, meningioma or pituitary adenoma, and in another embodiment of the present invention, The pharmaceutical composition inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
본 발명의 일 실시예에서, 서열번호 1의 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된 폴리펩티드, 또는 서열번호 1의 24번째 아미노산 Pro 내지 27번째 아미노산 Thr을 포함하고, 연속된 10개 이상의 아미노산을 포함하며, 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된, 폴리펩티드, 또는 서열번호 2, 4, 또는 5로 표시되는 아미노산 서열을 포함하는 폴리펩티드를 코딩하는 핵산 분자를 제공하고, 본 발명의 다른 실시예에서, 상기 핵산 분자는 서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환된 핵산 분자; 서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환되고, 61번째 핵산 내지 90번째 핵산 서열을 포함하는 핵산 분자; 서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환되고, 70번째 핵산 내지 99번째 핵산 서열을 포함하는 핵산 분자; 서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환되고, 52번째 핵산 내지 81번째 핵산 서열을 포함한다.In one embodiment of the present invention, a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with a non-phosphorylated amino acid, or a polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, and 10 or more consecutive amino acids Provides a nucleic acid molecule encoding a polypeptide, or a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 4, or 5, wherein the 25th amino acid Ser is substituted with a non-phosphorylated amino acid, and other embodiments of the present invention In the nucleic acid molecule, the 73rd to 75th nucleic acid of the nucleic acid sequence represented by SEQ ID NO: 3 is 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5' A nucleic acid molecule substituted with -GAG-3'; Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. a nucleic acid molecule that is substituted and comprises the 61st to 90th nucleic acid sequences; Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. a nucleic acid molecule that is substituted and comprises the 70th to 99th nucleic acid sequences; Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. is substituted and includes the 52nd to 81st nucleic acid sequences.
본 발명의 일 실시예에서, 상기의 핵산 분자를 포함하는 재조합 벡터를 제공한다. In one embodiment of the present invention, a recombinant vector containing the above nucleic acid molecule is provided.
본 발명의 일 실시예에서, 상기의 재조합 벡터를 포함하는 재조합 세포를 제공한다. In one embodiment of the present invention, a recombinant cell containing the above recombinant vector is provided.
본 발명의 일 실시예에서는 대상체의 암세포에서 서열번호 1로 표시되는 FoxM1 단백질 중 25번째 아미노산 Ser이 인산화되었거나 Asp 또는 Glu으로 치환된 경우, 암의 전이 가능성이 높은 것으로 판단하는 단계를 포함하는, 암의 전이 위험성 판단을 위한 정보제공방법을 제공한다. In one embodiment of the present invention, when the 25th amino acid Ser of the FoxM1 protein shown in SEQ ID NO: 1 in the subject's cancer cells is phosphorylated or substituted with Asp or Glu, the cancer metastasis process is determined to be highly likely. Provides an information provision method for determining the risk of metastasis.
본 발명의 다른 실시예에서는 대상체의 암세포에서 서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환된 경우, 암의 전이 가능성이 높은 것으로 판단하는 단계를 포함하는, 암의 전이 위험성 판단을 위한 정보제공방법을 제공한다. In another embodiment of the present invention, the 73rd to 75th nucleic acid of the nucleic acid sequence represented by SEQ ID NO: 3 in the subject's cancer cells is 5'-GAT-3', 5'-GAC-3', 5'-GAA-3. ', or 5'-GAG-3', provides a method of providing information for determining the risk of cancer metastasis, including the step of determining that the possibility of cancer metastasis is high.
본 발명의 일 실시예에서, 서열번호 1의 25번째 아미노산 Ser이 Asp, 또는 Glu로 치환된 폴리펩티드를 발현하는, 전이성 암을 연구하는 데에 이용될 수 있는 재조합 전이성 암 세포를 제공한다. In one embodiment of the present invention, a recombinant metastatic cancer cell that expresses a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with Asp or Glu, which can be used to study metastatic cancer, is provided.
본 발명의 일 실시예에서, 서열번호 3으로 표시되는 핵산 서열 중 73번째 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환된 핵산을 포함하는, 전이성 암을 연구하는 데에 이용될 수 있는 재조합 전이성 암세포를 제공한다. In one embodiment of the present invention, the 73rd to 75th nucleic acid of the nucleic acid sequence represented by SEQ ID NO: 3 is 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'. Provided are recombinant metastatic cancer cells that can be used to study metastatic cancer, comprising nucleic acids substituted with '-GAG-3'.
본 발명의 일 실시예에서, FOXM1 단백질을 코딩하는 유전자 또는 mRNA에 상보적으로 결합하여 FOXM1 단백질의 발현을 저하시키는 핵산분자를 포함하는, 암 치료용 약학 조성물을 제공한다. 본 발명의 일 실시예에서, 상기 FOXM1 단백질을 코딩하는 유전자 또는 mRNA는 서열번호 6으로 표시되는 핵산 서열을 포함하고, 본 발명의 다른 실시예에서, 상기 핵산분자는 서열번호 6의 187 내지 207번째 핵산 또는 709 내지 729번째 핵산에 특이적으로 결합하는 핵산 서열을 포함하며, 본 발명의 또 다른 실시예에서, 상기 핵산분자는 서열번호 7 내지 10 중 어느 하나의 핵산 서열을 포함하고, 본 발명의 또 다른 실시예에서, 상기 핵산분자는 shRNA, siRNA, 안티센스 RNA, 안티센스 DNA, 키메라 안티센스 DNA/RNA, miRNA, 및 라이보자임으로 구성된 군으로부터 선택된 어느 하나이고, 본 발명의 또 다른 실시예에서, 상기 암은 FOXM1 단백질을 과발현하거나, 25번째 아미노산 Ser이 Asp, 또는 Glu로 치환된 FOXM1 단백질을 발현하는 암이고, 본 발명의 또 다른 실시예에서, 상기 암은 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 또는 뇌하수체 선종이고, 본 발명의 또 다른 실시예에서, 상기 약학 조성물은 암세포의 사멸을 유도하거나, 암세포의 성장성, 이동성, 침습성, 및 전이성으로 구성된 군으로부터 선택된 하나 이상을 억제한다. In one embodiment of the present invention, a pharmaceutical composition for treating cancer is provided, comprising a nucleic acid molecule that reduces expression of FOXM1 protein by binding complementary to a gene or mRNA encoding FOXM1 protein. In one embodiment of the present invention, the gene or mRNA encoding the FOXM1 protein includes a nucleic acid sequence represented by SEQ ID NO: 6, and in another embodiment of the present invention, the nucleic acid molecule is 187 to 207 of SEQ ID NO: 6. It contains a nucleic acid sequence that specifically binds to a nucleic acid or nucleic acid positions 709 to 729, and in another embodiment of the present invention, the nucleic acid molecule comprises a nucleic acid sequence of any one of SEQ ID NOs: 7 to 10, and the nucleic acid molecule of the present invention In another embodiment, the nucleic acid molecule is any one selected from the group consisting of shRNA, siRNA, antisense RNA, antisense DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme, and in another embodiment of the present invention, the The cancer is a cancer that overexpresses the FOXM1 protein or expresses a FOXM1 protein in which the 25th amino acid Ser is replaced with Asp or Glu. In another embodiment of the present invention, the cancer is bone cancer, blood cancer, lung cancer, small cell lung cancer, Non-small cell lung cancer, squamous cell carcinoma, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate Cancer, uterine cancer, endometrial cancer, sarcoma, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, Renal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasm of the central nervous system (CNS), neuroectodermal cancer, spinal tumor, glioma, meningioma or pituitary adenoma, in another embodiment of the present invention. , the pharmaceutical composition induces death of cancer cells or inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
본 발명의 일 실시예에서, 서열번호 7 내지 10 중 어느 하나의 핵산 서열을 포함하고, shRNA, siRNA, 안티센스 RNA, 안티센스 DNA, 키메라 안티센스 DNA/RNA, miRNA, 및 라이보자임으로 구성된 군으로부터 선택된 어느 하나이고, 본 발명의 일 실시예에서, 상기 핵산 분자는 FOXM1 단백질을 코딩하는 유전자 또는 mRNA에 상보적으로 결합하고, 본 발명의 다른 실시예에서, 서열번호 1의 187 내지 207번째 핵산 또는 709 내지 729번째 핵산에 특이적으로 결합한다. In one embodiment of the present invention, any nucleic acid sequence comprising any one of SEQ ID NOs: 7 to 10 and selected from the group consisting of shRNA, siRNA, antisense RNA, antisense DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme One, in one embodiment of the present invention, the nucleic acid molecule binds complementary to a gene or mRNA encoding FOXM1 protein, and in another embodiment of the present invention, the nucleic acid 187 to 207 or 709 to 709 of SEQ ID NO: 1 Binds specifically to nucleic acid number 729.
본 발명의 일 실시예에서, 서열번호 7 내지 10 중 어느 하나의 핵산 서열을 포함하는 재조합 바이러스 벡터를 제공하고, 본 발명의 다른 실시예에서, 상기 재조합 바이러스 벡터는 shRNA, siRNA, 안티센스 RNA, 안티센스 DNA, 키메라 안티센스 DNA/RNA, miRNA, 및 라이보자임으로 구성된 군으로부터 선택된 어느 하나를 발현한다. In one embodiment of the present invention, a recombinant viral vector is provided comprising any one nucleic acid sequence of SEQ ID NOs: 7 to 10, and in another embodiment of the present invention, the recombinant viral vector is shRNA, siRNA, antisense RNA, antisense Expresses any one selected from the group consisting of DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme.
본 발명의 일 실시예에서, 상기 약학 조성물; 핵산 분자; 또는 재조합 바이러스 벡터를 암 치료가 필요한 대상체에게 투여하는 단계를 포함하는, 암 치료 방법을 제공한다.In one embodiment of the present invention, the pharmaceutical composition; nucleic acid molecule; Alternatively, a cancer treatment method is provided, comprising administering a recombinant viral vector to a subject in need of cancer treatment.
본 발명의 FoxM1 점 돌연변이를 포함하는 단백질 및/또는 펩티드 또는 shRNA는 암세포의 비정상적인 성장에 의한 각종 질환, 특히 일차성 및 전이성 고형암과 백혈병 등의 같은 암 질환 치료에 유용하게 사용될 수 있다.The protein and/or peptide or shRNA containing the FoxM1 point mutation of the present invention can be usefully used in the treatment of various diseases caused by abnormal growth of cancer cells, especially cancer diseases such as primary and metastatic solid cancer and leukemia.
도 1은 FoxM1과 PLK1의 발현이 비소세포폐암 환자 중 선암 환자에서 그 발현과 환자 생존율의 관계를 분석한 결과이다.Figure 1 shows the results of analyzing the relationship between the expression of FoxM1 and PLK1 in adenocarcinoma patients among non-small cell lung cancer patients and patient survival rate.
A: cBioPortal에서 빅데이터를 이용해 FoxM1과 PLK1이 폐암에서의 mRNA 발현 상관관계를 Spearman 과 Pearson으로 분석한 그래프이다.A: This is a graph analyzing the correlation between FoxM1 and PLK1 mRNA expression in lung cancer using big data from cBioPortal using Spearman and Pearson.
B: NSCLC 환자 mRNA 발현에서 PLK1과 FoxM1 사이의 상관관계를 cBio-Portal Spearman's coefficient로 분석한 결과이다.B: This is the result of analyzing the correlation between PLK1 and FoxM1 in NSCLC patient mRNA expression using cBio-Portal Spearman's coefficient.
C: NSCLC 환자 mRNA 발현에서 PLK1과 FoxM1 사이의 상관관계를 cBio-Portal Pearson's coefficient로 분석한 결과이다.C: This is the result of analyzing the correlation between PLK1 and FoxM1 in NSCLC patient mRNA expression using cBio-Portal Pearson's coefficient.
D: KM PLOTTER 분석을 통해서 폐암환자들의 생존율을 PLK1과 FoxM1 발현에 따라 분석한 그래프이다.D: This is a graph analyzing the survival rate of lung cancer patients according to PLK1 and FoxM1 expression through KM PLOTTER analysis.
E: KM PLOTTER 분석을 통해서 전이성 폐암에서 폐암환자들의 생존율을 PLK1과 FoxM1 발현에 따라 분석한 그래프이다.E: This is a graph analyzing the survival rate of lung cancer patients with metastatic lung cancer according to the expression of PLK1 and FoxM1 through KM PLOTTER analysis.
F: KM PLOTTER 분석을 통해서 전이성 폐암 진행단계에 따른 폐암환자들의 생존율을 PLK1과 FoxM1 발현에 따라 분석한 그래프이다.F: This is a graph analyzing the survival rate of lung cancer patients according to the stage of metastatic lung cancer according to the expression of PLK1 and FoxM1 through KM PLOTTER analysis.
G: 전이성 폐암 진행단계에 따른 폐암환자들에서 상피간엽이행 마커들의 발현을 Heatmap 분석을 통해서 보여준 그래프이다.G: This is a graph showing the expression of epithelial-mesenchymal transition markers in lung cancer patients according to the stage of metastatic lung cancer through heatmap analysis.
도 2는 전이성 폐암세포에서 FoxM1과 활성형 PLK1의 임상적 연관성 분석한 결과이다.Figure 2 shows the results of analyzing the clinical correlation between FoxM1 and active PLK1 in metastatic lung cancer cells.
A: TGF-β를 처리한 폐암세포 A549에서 FoxM1, PLK1 그리고 상피간엽이행 마커들의 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.A: This is a graph showing the mRNA expression of FoxM1, PLK1, and epithelial-mesenchymal transition markers in lung cancer cells A549 treated with TGF-β using real-time polymerase chain reaction (Real-time PCR).
B: TGF-β를 처리한 폐암세포 H358에서 FoxM1, PLK1 그리고 상피간엽이행 마커들의 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.B: This is a graph showing the mRNA expression of FoxM1, PLK1, and epithelial-mesenchymal transition markers in lung cancer cells H358 treated with TGF-β using real-time polymerase chain reaction (Real-time PCR).
C: TGF-β를 처리한 폐암세포 H460에서 FoxM1, PLK1 그리고 상피간엽이행 마커들의 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.C: This is a graph showing the mRNA expression of FoxM1, PLK1, and epithelial-mesenchymal transition markers in lung cancer cells H460 treated with TGF-β using real-time polymerase chain reaction (Real-time PCR).
D: TGF-β를 처리한 폐암세포 A549, H358, H460에서 FoxM1, PLK1 그리고 상피간엽이행 마커들의 단백질 발현을 면역블롯법으로 관찰한 결과이다.D: This is the result of observing the protein expression of FoxM1, PLK1, and epithelial-mesenchymal transition markers in lung cancer cells A549, H358, and H460 treated with TGF-β using immunoblotting.
E: A549 세포에 5 ng/ml TGF-b를 처리한 후 phosphatase (CIP) 처리에 의해서 FoxM1과 PLK1의 인산화의 감소를 관찰한 결과이다.E: This is the result of observing a decrease in phosphorylation of FoxM1 and PLK1 by phosphatase (CIP) treatment after treating A549 cells with 5 ng/ml TGF-b.
F: H460 세포에 5 ng/ml TGF-b를 처리한 후 phosphatase (CIP) 처리에 의해서 FoxM1과 PLK1의 인산화의 감소를 관찰한 결과이다.F: This is the result of observing a decrease in phosphorylation of FoxM1 and PLK1 by phosphatase (CIP) treatment after treating H460 cells with 5 ng/ml TGF-b.
도 3은 PLK1 활성형에 의한 FoxM1의 인산화 및 인산화 부위를 분석한 결과이다.Figure 3 shows the results of analysis of phosphorylation and phosphorylation sites of FoxM1 by PLK1 active form.
A: Myc 표지된 FoxM1에서 Myc항체를 이용하여 면역침강법을 진행하여 FoxM1과 PLK1이 상호작용함을 관찰한 결과이다.A: This is the result of observing the interaction between FoxM1 and PLK1 by performing immunoprecipitation using Myc antibody on Myc-labeled FoxM1.
B : A549 세포에 5 ng/ml TGF-b를 처리한 조건에서 PLK1 항체를 이용하여 면역침강법을 진행하여 FoxM1과 PLK1이 상호작용함을 관찰한 결과이다.B: This is the result of observing the interaction between FoxM1 and PLK1 by performing immunoprecipitation using a PLK1 antibody in A549 cells treated with 5 ng/ml TGF-b.
C : H460 세포에 5 ng/ml TGF-b를 처리한 조건에서 PLK1 항체를 이용하여 면역침강법을 진행하여 FoxM1과 PLK1이 상호작용함을 관찰한 결과이다.C: This is the result of observing the interaction between FoxM1 and PLK1 by performing immunoprecipitation using a PLK1 antibody in H460 cells treated with 5 ng/ml TGF-b.
D: GST가 표지된 FoxM1을 이용하여 활성형 PLK1(PLK1 TD)와 함께 인산화 효소 반응법을 수행하여 활성형 PLK1이 FoxM1을 인산화 시킴을 관찰한 결과이다.D: This is the result of performing a phosphorylase reaction using GST-labeled FoxM1 with active PLK1 (PLK1 TD) and observing that active PLK1 phosphorylates FoxM1.
E: 액체크로마토그래피 질량분석법을 통해서 PLK1에 의한 인산화된 FoxM1 인산화 가능 후보 부위를 확인한 결과이다.E: This is the result of confirming the candidate site for phosphorylation of FoxM1 phosphorylated by PLK1 through liquid chromatography mass spectrometry.
F: 부분 특이적 돌연변이 치환법을 이용하여 FoxM1의 인산화 후보 부위를 알라닌으로 치환한 비인산화 점 돌연변이체 단백질과 활성형 PLK1을 이용하여 인산화 효소 반응법을 수행한 결과이다.F: This is the result of a phosphorylation enzyme reaction using a non-phosphorylated point mutant protein in which the phosphorylation candidate site of FoxM1 was replaced with alanine using a partial-specific mutation substitution method and the active form of PLK1.
G: FoxM1의 인산화 후보 부위를 알라닌으로 치환한 비인산화 점 돌연변이체 단백질과 활성형 PLK1을 이용하여 인산화 효소 반응법을 수행한 결과이다.G: This is the result of a phosphorylation enzyme reaction using a non-phosphorylated point mutant protein in which the phosphorylation candidate site of FoxM1 was replaced with alanine and the active form of PLK1.
H: 여러종에서 FoxM1 인산화 위치를 표시한 결과이다. H: Results showing FoxM1 phosphorylation sites in several species.
도 4는 폐암세포 A549에서 FoxM1의 인산화 및 비인산화 점 돌연변이체의 과발현이 암세포의 이동성과 침습성에 미치는 영향을 측정한 실험이다.Figure 4 is an experiment measuring the effect of overexpression of phosphorylated and non-phosphorylated point mutants of FoxM1 in lung cancer cell A549 on the mobility and invasiveness of cancer cells.
A : 폐암세포 A549에 FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 후 FoxM1 단백질 과발현 유무와 중간엽이행 마커(N-cadherin) 변화를 면역블롯으로 관찰한 결과이다.A: This is the result of observing FoxM1 protein overexpression and changes in mesenchymal transition marker (N-cadherin) by immunoblot after expressing FoxM1 phosphorylated and non-phosphorylated point mutants in lung cancer cell A549.
B : 폐암세포 A549에 FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 후 FoxM1 mRNA 과발현 유무와 중간엽이행 마커(N-cadherin) mRNA 변화를 연쇄반응(Real-time PCR)을 통해 관찰한 결과이다.B: This is the result of observing FoxM1 mRNA overexpression and changes in mesenchymal transition marker (N-cadherin) mRNA through chain reaction (real-time PCR) after expressing FoxM1 phosphorylated and non-phosphorylated point mutants in lung cancer cell A549.
C : 폐암세포 A549에 FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 후 각각의 점 돌연변이체를 발현하는 세포의 세포증식 정도를 시간별로 관찰한 결과이다.C: This is the result of expressing FoxM1 phosphorylated and non-phosphorylated point mutants in lung cancer cell A549, and then observing the cell proliferation of cells expressing each point mutant over time.
D : FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포 A549에서 인서트(inset)를 이용한 이동성 실험(migration assay)을 통해서 세포의 이동성 양상을 현미경으로 관찰한 결과이다.D: This is the result of observing the cell migration pattern under a microscope through a migration assay using an insert in lung cancer cells A549 expressing FoxM1 phosphorylation and non-phosphorylation point mutants.
E : FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포 A549에서 matrigel과 인서트(inset)를 이용한 침습성 실험(Invasion assay)을 통해서 세포의 침습성 양상을 관찰한 결과이다.E: This is the result of observing cell invasiveness through an invasion assay using matrigel and insert in lung cancer cells A549 expressing FoxM1 phosphorylation and non-phosphorylation point mutants.
F : FoxM1 인산화 점 돌연변이체와 3개 점 동시 돌연변이체를 발현시킨 폐암세포 A549에서 상처 치유 분석 실험(Wound healing assay)을 통해서 세포의 이동성 양상을 관찰한 결과이다.F: This is the result of observing cell mobility through a wound healing assay in lung cancer cells A549 expressing a FoxM1 phosphorylation point mutant and three point simultaneous mutants.
G : FoxM1 인산화 점 돌연변이체와 3개 점 동시 돌연변이체를 발현시킨 폐암세포 A549에서 상처 치유 분석 실험(Wound healing assay)을 통해 72h 때 치유 (Healing) 상대적 거리를 막대 그래프로 나타냈다.G: The relative healing distance at 72h was shown in a bar graph through a wound healing assay in lung cancer cells A549 expressing a FoxM1 phosphorylation point mutant and three point co-mutants.
도 5는 동물모델에서 FoxM1의 인산화 및 비인산화 점 돌연변이체를 과발현하는 암세포의 전이성과 종양 형성능을 측정한 실험이다.Figure 5 is an experiment measuring the metastatic and tumorigenic ability of cancer cells overexpressing phosphorylated and non-phosphorylated point mutants of FoxM1 in an animal model.
A : FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스의 폐에 전이성 암생성 및 억제효과를 관찰한 결과이다.A: This is the result of observing the creation and inhibition of metastatic cancer in the lungs of mice when lung cancer cells expressing FoxM1 phosphorylation and non-phosphorylation point mutants were injected intravenously.
B : FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스의 폐에 전이되어 생성한 암조직을 H&E 염색하여 각 실험군의 암생성 및 억제효과를 관찰한 결과이다.B: This is the result of H&E staining of the cancer tissue that metastasized to the lungs of mice when lung cancer cells expressing FoxM1 phosphorylation and non-phosphorylation point mutants were injected intravenously, and the cancer formation and inhibition effects of each experimental group were observed.
C : FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스의 폐에 전이되어 생성한 암조직을 Ki-67 염색하여 각 실험군의 암생성 및 억제효과를 관찰한 결과이다.C: This is the result of observing the cancer formation and inhibitory effects of each experimental group by staining the cancer tissue that metastasized to the lungs of mice and stained with Ki-67 when lung cancer cells expressing FoxM1 phosphorylated and non-phosphorylated point mutants were injected intravenously.
D : FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스에서 얻은 폐조직을 분쇄하여 상피간엽이행 단백질 마커와 면역회피 인자 단백질의 변화를 관찰한 결과이다.D: This is the result of pulverizing lung tissue obtained from mice and observing changes in epithelial-mesenchymal transition protein markers and immune evasion factor proteins when lung cancer cells expressing FoxM1 phosphorylated and non-phosphorylated point mutants were injected intravenously.
E : FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스에서 얻은 폐조직을 분쇄하여 상피간엽 마커와 면역회피 인자 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.E: When lung cancer cells expressing FoxM1 phosphorylation and non-phosphorylation point mutants were injected intravenously, lung tissue obtained from mice was pulverized and the expression of epithelial-mesenchymal markers and immune evasion factor mRNA was analyzed by real-time polymerase chain reaction (Real-time PCR). This is a graph shown using:
F: FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포에서 caspase-3 효소의 활성을 측정하여 세포의 사멸정도를 관한 결과이다.F: Results of the degree of cell death by measuring the activity of caspase-3 enzyme in lung cancer cells expressing FoxM1 phosphorylation and non-phosphorylation point mutants.
도 6는 FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포와 공동배양한 monocyte THP-1세포의 종양관련 대식세포 (TAM)으로 분화에 미치는 효과를 관찰한 결과이다.Figure 6 shows the results of observing the effect on differentiation into tumor-associated macrophages (TAM) of monocyte THP-1 cells co-cultured with lung cancer cells expressing point mutants of the FoxM1 protein.
A: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포와 monocyte THP-1 세포를 공동배양하여 THP-1 세포에서 M1, M2 마커의 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.A: Lung cancer cells expressing point mutants of the FoxM1 protein were co-cultured with monocyte THP-1 cells, and mRNA expression of M1 and M2 markers in THP-1 cells was measured using real-time polymerase chain reaction (Real-time PCR). This is the graph shown.
B: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포와 monocyte THP-1 세포를 공동배양하여 A549 세포에서 M2 유도 인자와 면역회피 인자 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.B: Lung cancer cells expressing a point mutant of the FoxM1 protein were co-cultured with monocyte THP-1 cells, and M2-inducing factor and immune evasion factor mRNA expression was measured in A549 cells using real-time polymerase chain reaction (Real-time PCR). This is the graph shown.
C: FoxM1 단백질의 점 돌연변이체를 발현하는 배양 배지에서 ELISA 실험을 통하여 TGFB1, VEGFA 발현을 관찰한 결과이다.C: Results of observing the expression of TGFB1 and VEGFA through ELISA experiments in culture media expressing point mutants of the FoxM1 protein.
D: FoxM1 Knockdown 한 돌연변이 암세포와 THP-1 공동배양 하여 THP-1 세포에서 M2 마커의 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.D: This is a graph showing the mRNA expression of the M2 marker in THP-1 cells co-cultured with FoxM1 knockdown mutant cancer cells and THP-1 using real-time polymerase chain reaction (Real-time PCR).
E: FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스의 폐에 전이되어 생성한 폐조직을 TAM-마커인 CD68, CD163 염색하여 관찰한 결과이다.E: This is the result of observation of lung tissue that metastasized to the lungs of mice when lung cancer cells expressing FoxM1 phosphorylated and non-phosphorylated point mutants were injected intravenously and stained with TAM-markers CD68 and CD163.
F: FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스의 폐에 전이되어 생성한 폐조직을 TAM-마커인 CD68, CD163 염색한 결과를 수치화한 그래프이다.F: This is a graph quantifying the results of TAM-marker CD68 and CD163 staining of lung tissue produced by metastasis to the lungs of mice when lung cancer cells expressing FoxM1 phosphorylated and non-phosphorylated point mutants were injected intravenously.
G: FoxM1 인산화 및 비인산화 점 돌연변이체를 발현시킨 폐암세포를 정맥주사했을 때 마우스의 폐에 전이되어 생성한 폐조직을 분쇄하여 TAM-마커인 CD68, CD163 단백질의 변화를 관찰한 결과이다.G: This is the result of observing changes in TAM-marker CD68 and CD163 proteins by pulverizing lung tissue that metastasized to the lungs of mice when lung cancer cells expressing FoxM1 phosphorylated and non-phosphorylated point mutants were injected intravenously.
도 7는 FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포가 공동배양한 monocyte THP-1세포 및 T cell인 Jurkat 세포를 분화시켜 종양세포 면역회피에 미치는 효과를 관찰한 결과이다.Figure 7 shows the results of observing the effect on tumor cell immune evasion by differentiating monocyte THP-1 cells and Jurkat cells, a T cell, co-cultured with lung cancer cells expressing a point mutant of the FoxM1 protein.
A: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포와 monocyte THP-1 세포를 비율로 공동배양하여 폐암세포 생존율을 보여준 그래프이다.A: This is a graph showing the survival rate of lung cancer cells by co-culturing lung cancer cells expressing point mutants of the FoxM1 protein and monocyte THP-1 cells.
B: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포와 monocyte THP-1 세포를 공동배양하여 THP-1 세포에서 면역회피 인자 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.B: Graph showing immune evasion factor mRNA expression in THP-1 cells by co-culturing lung cancer cells expressing point mutants of the FoxM1 protein and monocyte THP-1 cells using real-time polymerase chain reaction (Real-time PCR). am.
C: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포에서 면역회피 인자 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.C: This is a graph showing the expression of immune evasion factor mRNA in lung cancer cells expressing point mutants of the FoxM1 protein using real-time polymerase chain reaction (Real-time PCR).
D: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포와 T 세포인 Jurkat 세포를 비율로 공동배양하여 폐암세포 생존율을 보여준 그래프이다.D: This is a graph showing the survival rate of lung cancer cells by co-culturing lung cancer cells expressing point mutants of the FoxM1 protein and Jurkat cells, which are T cells.
E: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포와 T 세포인 Jurkat 세포를 비율로 공동배양하여 Jurkat 세포에서 T 조절세포 마커 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.E: Lung cancer cells expressing point mutants of the FoxM1 protein and Jurkat cells, which are T cells, were co-cultured in proportion, and the expression of T regulatory cell marker mRNA in Jurkat cells was shown using real-time polymerase chain reaction (Real-time PCR). It's a graph.
F: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포에서 면역회피 인자와 M2 유도 인자 mRNA 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 이용하여 보여준 그래프이다.F: This is a graph showing the expression of immune evasion factor and M2 inducing factor mRNA in lung cancer cells expressing point mutants of the FoxM1 protein using real-time polymerase chain reaction (Real-time PCR).
G: FoxM1 단백질의 점 돌연변이체를 발현하는 폐암세포, monocyte THP-1 세포, T 세포인 Jurkat 세포를 비율로 공동배양하여 폐암세포 생존율을 보여준 그래프이다.G: This is a graph showing the survival rate of lung cancer cells by co-culturing lung cancer cells expressing point mutants of the FoxM1 protein, monocyte THP-1 cells, and Jurkat cells, which are T cells.
도 8는 FoxM1 비인산화 점 돌연변이체 펩티드를 제작하여 폐암세포에서의 세포사멸효과와 전이성을 평가를 통해서 펩티드 효능평가를 관찰한 결과이다.Figure 8 shows the results of observing the efficacy of the peptide by producing a FoxM1 non-phosphorylated point mutant peptide and evaluating its apoptosis effect and metastatic properties in lung cancer cells.
A : FoxM1 비인산화 점 돌연변이체 펩티드 서열이다.A: FoxM1 non-phosphorylated point mutant peptide sequence.
B: A549 세포에서 3종류의 FoxM1 비인산화 점 돌연변이체 펩티드의 세포사멸성의 효능을 관찰한 결과이다.B: Results of observing the apoptotic efficacy of three types of FoxM1 non-phosphorylated point mutant peptides in A549 cells.
C: A549세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 3종류의 FoxM1 비인산화 점 돌연변이체 펩티드(5μM FoxM1-S25A peptide)처리에 의해 암세포의 전이성 억제효능을 관찰한 결과이다. C: This is the result of observing the metastatic inhibition effect of cancer cells by treatment with three types of FoxM1 non-phosphorylated point mutant peptides (5 μM FoxM1-S25A peptide) under overexpression conditions of phosphorylated point mutants of FoxM1 in A549 cells.
도 9는 여러 암종에서 FoxM1 발현에 따른 환자의 생존을 분석한 결과이다.Figure 9 shows the results of analyzing patient survival according to FoxM1 expression in various carcinomas.
A : FoxM1의 발현에 따른 12개의 암종에 따른 환자의 생존율을 분석한 그래프이다.A: This is a graph analyzing the survival rate of patients in 12 types of carcinoma according to the expression of FoxM1.
B: 고형암에서 3종류의 FoxM1 비인산화 점 돌연변이체 펩티드의 세포사멸성의 효능을 관찰한 결과이다.B: This is the result of observing the apoptotic efficacy of three types of FoxM1 non-phosphorylated point mutant peptides in solid cancer.
도 10는 FoxM1 비인산화 점 돌연변이체 펩티드를 제작하여 폐암세포에서의 전이성과 종양관련 대식세포 분화성과 종양세포 면역회피능에 미치는 효과를 관찰한 결과이다.Figure 10 shows the results of producing a FoxM1 non-phosphorylated point mutant peptide and observing its effects on metastatic properties in lung cancer cells, tumor-related macrophage differentiation, and tumor cell immune evasion ability.
A : 세포투과성을 관찰하기 위한 peptide의 C말단에 FITC(형광물질)을 연결한 FoxM1 비인산화 점 돌연변이체 펩티드 서열이다.A: This is a FoxM1 non-phosphorylated point mutant peptide sequence in which FITC (a fluorescent substance) is connected to the C terminus of the peptide to observe cell permeability.
B : A549 세포에서 FoxM1 비인산화 점 돌연변이체 펩티드의 세포투과 정도를 관찰한 결과이다.B: This is the result of observing the degree of cell penetration of FoxM1 non-phosphorylated point mutant peptide in A549 cells.
C : A549 세포에 5ng/ml TGF-β를 처리후 FoxM1 비인산화 점 돌연변이체 펩타드(5μM FoxM1-S25A peptide)처리에 의해 중간엽이행 마커 mRNA 변화를 관찰한 결과이다.C: This is the result of observing changes in mesenchymal transition marker mRNA by treating A549 cells with 5ng/ml TGF-β and then treating them with FoxM1 non-phosphorylated point mutant peptide (5μM FoxM1-S25A peptide).
D: A549 세포에 5ng/ml TGF-β를 처리 후 FoxM1 비인산화 점 돌연변이체 펩타드(5μM FoxM1-S25A peptide)처리에 의해 암세포의 이동성의 변화를 관찰한 결과이다.D: This is the result of observing changes in the mobility of cancer cells by treating A549 cells with 5ng/ml TGF-β and then treating them with FoxM1 non-phosphorylated point mutant peptide (5μM FoxM1-S25A peptide).
E: A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 FoxM1 비인산화 점 돌연변이체 펩타드(5μM FoxM1-S25A peptide)처리에 의해 중간엽이행 마커 mRNA 변화를 관찰한 결과이다.E: This is the result of observing mesenchymal transition marker mRNA changes by treatment with FoxM1 non-phosphorylated point mutant peptide (5μM FoxM1-S25A peptide) under overexpression conditions of phosphorylated point mutant of FoxM1 in A549 cells.
F: A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 FoxM1 비인산화 점 돌연변이체 펩타드(5μM FoxM1-S25A peptide)처리에 의해 암세포의 이동성의 변화를 관찰한 결과이다.F: This is the result of observing changes in the mobility of cancer cells by treatment with a non-phosphorylated FoxM1 point mutant peptide (5 μM FoxM1-S25A peptide) under overexpression conditions of a phosphorylated point mutant of FoxM1 in A549 cells.
G: A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 FoxM1 비인산화 점 돌연변이체 펩타드(5μM FoxM1-S25A peptide)처리에 의해 STAT1, VEGFA, c-fos, IL6, CD274 mRNA 변화를 관찰한 결과이다.G: Results of observing changes in STAT1, VEGFA, c-fos, IL6, and CD274 mRNA by treatment with FoxM1 non-phosphorylated point mutant peptide (5μM FoxM1-S25A peptide) under overexpression conditions of phosphorylated point mutant of FoxM1 in A549 cells. am.
도 11은 전이성 폐암세포에서 FoxM1과 활성형 PLK1의 임상적 연관성 분석한 결과이다.Figure 11 shows the results of analyzing the clinical correlation between FoxM1 and active PLK1 in metastatic lung cancer cells.
A : KEGG 2019 pathway 분석을 통해서 폐암세포 A549의 암전이 조건에서 활성형 PLK1이 발현되는 침습성 세포들의 관련 pathway를 분석한 그래프이다.A: This is a graph analyzing the related pathways of invasive cells in which active PLK1 is expressed in cancer metastasis conditions of lung cancer cell A549 through KEGG 2019 pathway analysis.
B: TGF-β를 처리한 폐암세포 A549에서 FoxM1과 상피간엽이행 마커들의 단백질 발현을 면역블롯법으로 관찰한 결과이다.B: This is the result of observing the protein expression of FoxM1 and epithelial-mesenchymal transition markers in lung cancer cells A549 treated with TGF-β using immunoblotting.
C: TGF-β를 처리한 폐암세포 H358에서 FoxM1과 상피간엽이행 마커들의 단백질 발현을 면역블롯법으로 관찰한 결과이다.C: This is the result of observing the protein expression of FoxM1 and epithelial-mesenchymal transition markers in lung cancer cells H358 treated with TGF-β using immunoblotting.
D: TGF-β를 처리한 폐암세포 H460에서 FoxM1과 상피간엽이행 마커들의 단백질 발현을 면역블롯법으로 관찰한 결과이다.D: This is the result of observing the protein expression of FoxM1 and epithelial-mesenchymal transition markers in lung cancer cells H460 treated with TGF-β using immunoblotting.
도 12는 PLK1 활성형에 의한 FoxM1의 원형 과발현이 암세포의 이동성과 침습성에 미치는 영향을 측정한 실험이다.Figure 12 is an experiment measuring the effect of circular overexpression of FoxM1 by activated PLK1 on the mobility and invasiveness of cancer cells.
A : 폐암세포 A549에 FoxM1 원형 발현시킨 후 FoxM1 단백질 과발현 유무와 EMT 마커 변화를 면역블롯으로 관찰한 결과이다.A: This is the result of observing the presence or absence of FoxM1 protein overexpression and changes in EMT markers by immunoblot after expressing the original form of FoxM1 in lung cancer cell A549.
B : 폐암세포 A549에 FoxM1 원형 발현시킨 후 FoxM1 단백질 과발현 유무와 EMT 마커 변화를 연쇄반응(Real-time PCR)을 통해 관찰한 결과이다.B: This is the result of observing FoxM1 protein overexpression and changes in EMT markers through chain reaction (real-time PCR) after expressing the original form of FoxM1 in lung cancer cells A549.
C: FoxM1 원형 발현시킨 폐암세포 A549에서 인서트(inset)를 이용한 이동성 실험(migration assay)을 통해서 세포의 이동성을 crystal violet 염색된 암세포의 정도(intensity)를 Odyssey infrared imaging system 분석장치를 이용하여 암세포 이동성 양상을 보여주는 그래프이다.C: Cell mobility was measured through a migration assay using an insert in lung cancer cell A549 with original FoxM1 expression. The intensity of crystal violet-stained cancer cells was measured using the Odyssey infrared imaging system analysis device. This is a graph showing the pattern.
D : FoxM1 원형 발현시킨 폐암세포 A549에서 matrigel과 인서트(inset)를 이용한 침습성 실험(Invasion assay)을 통해서 세포의 침습성 양상을 관찰한 결과이다.D: This is the result of observing the invasiveness of the cells through an invasion assay using matrigel and insert in lung cancer cells A549 that expressed the original FoxM1.
E : FoxM1 원형 발현시킨 폐암세포 A549에서 면역회피인자인 CD274의 발현을 연쇄반응(Real-time PCR)을 통해 관찰한 결과이다.E: This is the result of observing the expression of CD274, an immune evasion factor, in lung cancer cells A549 expressing the original FoxM1 through chain reaction (real-time PCR).
도 13은 FoxM1 원형 발현시킨 암세포와 인간 대식세포인 THP-1 세포 공동배양시 THP-1의 TAM 마커의 증가 및 분화를 관찰한 결과이다.Figure 13 shows the results of observing the increase and differentiation of the TAM marker of THP-1 when co-culturing cancer cells expressing the original FoxM1 and THP-1 cells, which are human macrophages.
A : FoxM1 원형 발현시킨 암세포와 인간 대식세포인 THP-1 세포 공동배양하여 THP-1 세포에서 M1, M2 마커의 발현을 관찰한 결과이다.A: This is the result of co-culturing cancer cells expressing the original FoxM1 and THP-1 cells, which are human macrophages, and observing the expression of M1 and M2 markers in THP-1 cells.
B: FoxM1 원형 발현시킨 암세포와 인간 대식세포인 THP-1 세포 공동배양하여 A549 세포에서 M2 유도 인자의 발현을 관찰한 결과이다.B: This is the result of observing the expression of M2-inducing factors in A549 cells by co-culturing cancer cells expressing the original FoxM1 and THP-1 cells, a human macrophage.
C: FoxM1 원형 발현시킨 암세포를 48시간 배양한 배지에서 TGFB1, VEGFA 발현을 ELISA 실험을 통하여 관찰한 결과이다.C: This is the result of observing the expression of TGFB1 and VEGFA in the medium in which cancer cells expressing the original FoxM1 were cultured for 48 hours through ELISA experiment.
D: FoxM1 원형 발현시킨 암세포 주입한 폐동물조직에서 대식세포 마커인 CD68와 종양관련대식세포 마커인 CD163 마커를 면역염색법을 이용하여 관찰한 결과이다.D: This is the result of observing CD68, a macrophage marker, and CD163, a tumor-related macrophage marker, in lung animal tissue injected with cancer cells expressing the original FoxM1 using immunostaining.
도 14는 폐암세포에서 FoxM1 mRNA 발현 억제를 위해 제작한 각각의 FoxM1 shRNA 처리하였을 때의 FoxM1발현억제 효과를 관찰한 결과이다.Figure 14 shows the results of observing the effect of suppressing FoxM1 expression when treated with each FoxM1 shRNA prepared to suppress FoxM1 mRNA expression in lung cancer cells.
A : 폐암세포 A549에 각각의 FoxM1 shRNA 처리로 FoxM1 발현억제 정도를 mRNA 발현 양상으로 보여주는 그래프이다. A: This is a graph showing the degree of inhibition of FoxM1 expression by treatment with each FoxM1 shRNA in lung cancer cell A549 in terms of mRNA expression pattern.
B : 폐암세포 A549에 각각의 FoxM1 shRNA 처리로 FoxM1 발현억제 정도를 단백질 발현 양상으로 보여주는 결과이다.B: Results showing the degree of inhibition of FoxM1 expression by treatment with each FoxM1 shRNA in lung cancer cell A549 in terms of protein expression pattern.
C : 폐암세포 A549에 FoxM1 shRNA 처리로 FoxM1 발현억제가 암세포의 이동성 억제효과를 주는지 24시간 간격으로 보여주는 결과이다.C: Results showing at 24-hour intervals whether suppression of FoxM1 expression by FoxM1 shRNA treatment on lung cancer cell A549 has an inhibitory effect on cancer cell mobility.
D : 폐암세포 A549에 FoxM1 shRNA 처리로 FoxM1 발현억제가 암세포의 이동성 억제효과를 주는지 72시간 때 효과를 보여주는 결과이다.D: Results showing the effect of suppressing FoxM1 expression by treating lung cancer cell A549 with FoxM1 shRNA at 72 hours to suppress the mobility of cancer cells.
E : 폐암세포 A549에 FoxM1 shRNA 처리로 FoxM1 발현억제가 TGF-β 처리에 의해서 유도되는 암세포 전이 환경에서 상피간엽이행 마커(CDH1, CDH2) 의 mRNA 발현 양상을 보여주는 그래프이다.E: This is a graph showing the mRNA expression pattern of epithelial-mesenchymal transition markers (CDH1, CDH2) in a cancer cell metastasis environment where suppression of FoxM1 expression by treatment of FoxM1 shRNA in lung cancer cell A549 is induced by TGF-β treatment.
F: 폐암세포 A549에 FoxM1 shRNA 처리에 의해 암세포의 사멸이 유도되는 것을 보여주는 그래프이다.F: This is a graph showing that cancer cell death is induced by FoxM1 shRNA treatment in lung cancer cell A549.
도 15는 FoxM1 원형 단백질이 발현되는 A549 폐암세포에서 FoxM1 mRNA 발현 억제 물질인 FoxM1 shRNA 처리하였을 때의 대식세포의 분화능을 관찰한 결과이다.Figure 15 shows the results of observing the differentiation ability of macrophages when A549 lung cancer cells expressing the FoxM1 circular protein were treated with FoxM1 shRNA, an inhibitor of FoxM1 mRNA expression.
A : FoxM1 원형 단백질이 발현되는 A549 폐암세포에서 FoxM1 mRNA 발현 억제 물질인 FoxM1 shRNA 처리하였을 때 FoxM1의 mRNA 발현을 보여주는 결과이다.A: This result shows the mRNA expression of FoxM1 when A549 lung cancer cells expressing the FoxM1 circular protein were treated with FoxM1 shRNA, an inhibitor of FoxM1 mRNA expression.
B : FoxM1 원형 단백질이 발현되는 A549 폐암세포에서 FoxM1 mRNA 발현 억제 물질인 FoxM1 shRNA 처리한 암세포와 인간 대식세포인 THP-1 세포 공동배양시 THP-1의 TAM 마커의 변화를 관찰한 결과이다. B: This is the result of observing changes in the TAM marker of THP-1 when co-culturing cancer cells treated with FoxM1 shRNA, an inhibitor of FoxM1 mRNA expression, and THP-1 cells, a human macrophage, in A549 lung cancer cells expressing the FoxM1 circular protein.
C : FoxM1 원형 단백질이 발현되는 A549 폐암세포에서 FoxM1 mRNA 발현 억제 물질인 FoxM1 shRNA 처리하였을 때 IFITM1의 mRNA 발현을 보여주는 결과이다.C: Results showing the mRNA expression of IFITM1 when A549 lung cancer cells expressing the FoxM1 circular protein were treated with FoxM1 shRNA, an inhibitor of FoxM1 mRNA expression.
도 16은 폐암세포에서 FoxM1 억제 물질인 Thiostrepton을 처리하였을 때의 중간엽이행 마커 대식세포 M2 유도 인자의 변화를 관찰한 결과이다.Figure 16 shows the results of observing changes in the mesenchymal transition marker macrophage M2-inducing factor when lung cancer cells are treated with Thiostrepton, a FoxM1 inhibitor.
A : 폐암세포 A549에 Thiostrepton 처리로 FoxM1의 기능 억제가 FoxM1 원형(WT)의 과발현에 의해서 유도되는 암세포 전이 환경에서 중간엽이행 마커(CDH2, VIM, SNAI1, SNAI2)의 mRNA 발현 양상을 보여주는 결과이다.A: This result shows the mRNA expression pattern of mesenchymal transition markers (CDH2, VIM, SNAI1, SNAI2) in a cancer cell metastasis environment where inhibition of FoxM1 function by treatment with Thiostrepton in lung cancer cell A549 is induced by overexpression of original FoxM1 (WT). .
B : 폐암세포 A549에 Thiostrepton 처리로 FoxM1의 기능 억제가 FoxM1 원형(WT)의 과발현에 의해 M2 분화로 유도 되는 조건에서 M2 유도인자 (IL6, VEGFA)와 면역관문 회피인자 (CD274)의 mRNA 발현 양상을 보여주는 결과이다.B: mRNA expression patterns of M2 inducers (IL6, VEGFA) and immune checkpoint evasion factor (CD274) under conditions in which inhibition of FoxM1 function by treatment with thiostrepton in lung cancer cell A549 induces M2 differentiation by overexpression of original FoxM1 (WT). This result shows.
도 17 내지 19는 야생형 FOXM1 유전자의 핵산 서열 (서열번호 3) 및 FoxM1 단백질의 아미노산 서열 (서열번호 1)을 나타낸 것이고, 도 20 내지 도 22는 서열번호 1 내지 11의 서열을 나타낸다: 서열번호 6은 접근번호 U74613의 인간 FOXM1B mRNA, 서열번호 7은 본 발명의 일 실시예에 따른 첫번째 타겟의 정방향 서열, 서열번호 8은 본 발명의 일 실시예에 따른 첫번째 타겟의 역방향 서열, 서열번호 9는 본 발명의 일 실시예에 따른 두번째 타겟의 정방향 서열, 서열번호 10은 본 발명의 일 실시예에 따른 두번째 타겟의 역방향 서열, 및 서열번호 11은 FOXM1 1B의 단백질 서열. Figures 17 to 19 show the nucleic acid sequence of the wild-type FOXM1 gene (SEQ ID NO: 3) and the amino acid sequence of the FoxM1 protein (SEQ ID NO: 1), and Figures 20 to 22 show the sequences of SEQ ID NOs: 1 to 11: SEQ ID NO: 6 is human FOXM1B mRNA with accession number U74613, SEQ ID NO: 7 is the forward sequence of the first target according to an embodiment of the present invention, SEQ ID NO: 8 is the reverse sequence of the first target according to an embodiment of the present invention, and SEQ ID NO: 9 is the present sequence. The forward sequence of the second target according to an embodiment of the invention, SEQ ID NO: 10 is the reverse sequence of the second target according to an embodiment of the present invention, and SEQ ID NO: 11 is the protein sequence of FOXM1 1B.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 통합된다.All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are hereby incorporated by reference.
본 발명의 발명자는 FoxM1과 PLK1의 발현이 비소세포폐암 환자 중 선암 환자에서 그 발현이 높으며 환자 생존율과 반비례 관계가 있어 예후에 있어 진단 마커로서의 활용할 수 있음을 확인하였다 (도 1).The inventor of the present invention confirmed that the expression of FoxM1 and PLK1 is high in adenocarcinoma patients among non-small cell lung cancer patients and has an inverse relationship with patient survival rate, so they can be used as diagnostic markers for prognosis (Figure 1).
본 발명자는 비소세포폐암에서 FoxM1과 PLK1의 상관관계를 분석하고 임상적인 유의성을 검토하기 위하여 cBioPortal에서 등록된 빅데이터를 이용하여 폐 선암 환자에서 FoxM1과 PLK1의 mRNA 발현간의 상관관계를 Spearman과 Pearson의 상관계수를 분석한 결과 유의적인 범위에서 두 인자의 발현에는 양의 상관관계가 있음을 확인하였다(도 1A). 뿐만 아니라 다른 세포주기에 관련된 인자들도 함께 그 상관성을 분석한 결과, FoxM1과 MKI67의 발현이 PLK1발현과 높은 상관계수를 보이는 인자로 분석되었다(도 1B, 1C). 다음으로는 비소세포폐암 환자에서 FOXM1과 PLK1이 임상적 연관성을 확인하기 위해서 FOXM1과 PLK1의 발현량에 따른 환자의 전체 생존율을 빅데이터를 분석을 통해서 확인하였다(도 1D, 1E, 1F). 비소세포폐암 환자에서 PLK1과 FOXM1 발현이 높은 환자의 생존율은 PLK1과 FOXM1의 발현이 낮은 환자의 생존율에 비해 현저히 낮은 것을 확인할 수 있었다(도 1D). 비소세포폐암은 폐 선암(adenocarcinoma)과 편평세포폐암 (Squamous lung cell carcinoma)으로 나누는 데, 이를 나누어서 분석한 결과에 따르면, 폐선암 환자에서 PLK1과 FOXM1 발현이 높은 환자의 생존율은 PLK1과 FOXM1의 발현이 낮은 환자의 생존율에 비해 현저히 낮은 것을 확인할 수 있었다(도 1E). 특히 폐선암환자 중 전이암이 관찰되는 stage 3-4인 환자 대상으로 재분석한 결과, PLK1과 FOXM1 발현이 높은 환자의 생존율은 PLK1과 FOXM1의 발현이 낮은 환자의 생존율에 비해 현저히 낮은 것을 확인할 수 있었다(도 1F). 추가적으로 폐선암 환자를 단계 별로 분류하고 FoxM1과 PLK1의 발현정도를 히트맵으로 분석한 결과, 정상 조직에 비해 암환자에서 그리고 1기보다는 2기 내지 4기의 조직에서 PLK1과 FOXM1 발현이 높음을 확인하였다(도 1G). In order to analyze the correlation between FoxM1 and PLK1 in non-small cell lung cancer and examine its clinical significance, the present inventor used big data registered in cBioPortal to determine the correlation between the mRNA expression of FoxM1 and PLK1 in lung adenocarcinoma patients using Spearman and Pearson's method. As a result of analyzing the correlation coefficient, it was confirmed that there was a positive correlation in the expression of the two factors in a significant range (Figure 1A). In addition, as a result of analyzing the correlation with other cell cycle-related factors, the expression of FoxM1 and MKI67 was analyzed as a factor showing a high correlation coefficient with PLK1 expression (Figures 1B, 1C). Next, in order to confirm the clinical correlation between FOXM1 and PLK1 in non-small cell lung cancer patients, the overall survival rate of patients according to the expression level of FOXM1 and PLK1 was confirmed through big data analysis (Figures 1D, 1E, 1F). In non-small cell lung cancer patients, the survival rate of patients with high expression of PLK1 and FOXM1 was confirmed to be significantly lower than that of patients with low expression of PLK1 and FOXM1 (Figure 1D). Non-small cell lung cancer is divided into adenocarcinoma and squamous lung cell carcinoma. According to the results of the analysis, the survival rate of patients with high PLK1 and FOXM1 expression in lung adenocarcinoma patients is higher than that of lung adenocarcinoma. It was confirmed that the survival rate was significantly lower than the low patient survival rate (Figure 1E). In particular, as a result of re-analysis of patients with stage 3-4 lung adenocarcinoma where metastatic cancer was observed, it was confirmed that the survival rate of patients with high PLK1 and FOXM1 expression was significantly lower than the survival rate of patients with low expression of PLK1 and FOXM1. (Figure 1F). Additionally, as a result of classifying lung adenocarcinoma patients by stage and analyzing the expression levels of FoxM1 and PLK1 using a heat map, it was confirmed that PLK1 and FOXM1 expression was higher in cancer patients compared to normal tissues and in stage 2 to 4 tissues rather than stage 1. (Figure 1G).
따라서 비소세포폐암 중 폐선암에서 PLK1 및 FOXM1 발현량과 환자의 생존율의 연관성을 확인할 수 있었으며, 특히 PLK1과 FOXM1 발현이 높은 경우에 환자의 생존율을 낮추는 인자임을 알 수 있었다. 또한 암전이가 많이 진행된 환자에서 PLK1과 FOXM1 발현량이 높게 발현되어 환자의 예후를 알 수 있는 진단 마커로서의 가치를 인식할 수 있었다.Therefore, we were able to confirm the correlation between PLK1 and FOXM1 expression levels and patient survival rate in lung adenocarcinoma among non-small cell lung cancers. In particular, it was found that high PLK1 and FOXM1 expression was a factor that lowered patient survival rate. In addition, in patients with advanced cancer metastasis, the expression levels of PLK1 and FOXM1 were high, indicating their value as diagnostic markers to determine the patient's prognosis.
본 발명의 발명자는 FoxM1과 PLK1의 발현이 비소세포폐암 세포에서 암 전이가 유도되는 환경에서 그 발현이 높으며 PLK1 활성 증가 및 FoxM1의 인산화와 비례 관계가 있어 암전이와의 상관관계를 확인하였다 (도 2).The inventors of the present invention confirmed the correlation with cancer metastasis because the expression of FoxM1 and PLK1 is high in an environment where cancer metastasis is induced in non-small cell lung cancer cells and is proportional to the increase in PLK1 activity and phosphorylation of FoxM1 (Figure 2 ).
비소세포폐암 환자를 대상으로 FoxM1과 PLK1의 발현과 생존율, 암 단계에 따른 발현분석에서 FoxM1과 PLK1의 발현이 전이암에서 높아 생존율을 낮추는 원인으로 작용할 가능성이 임상데이터에서 관찰된 바, 본 발명자들은 전이암 모델에서 FoxM1과 PLK1의 기능을 관찰하고자 하였다. In the analysis of the expression and survival rate of FoxM1 and PLK1 in patients with non-small cell lung cancer, and expression according to cancer stage, the present inventors observed in clinical data that the expression of FoxM1 and PLK1 is high in metastatic cancer, which may lower the survival rate. We sought to observe the functions of FoxM1 and PLK1 in a metastatic cancer model.
이를 위하여 TGF-β를 처리하여 암전이 세포모델인 상피간엽이행(EMT)을 유도하는 과정에서 PLK1과 FoxM1의 발현 변화와 PLK1의 활성화여부를 관찰하고자 비소세포폐암 세포주 A549, NCI-H358, 및 NCI-H460 세포주에 각각 TGF-β를 처리하여 상피간엽이행을 유도한 후 mRNA 발현량과 단백질 발현량을 분석하였다. 먼저 TGF-β를 처리한 군에서 간엽형질 마커인 CDH2, SNAl1 SNAI2의 mRNA 발현량 증가와 상피형질 마커인 CDH1의 mRNA 발현량 감소를 관찰하였다. 이 조건에서 대조군에 비하여 PLK1과 FOXM1의 mRNA 발현량의 증가를 관찰할 수 있었다(도 2A, 2B, 2C). 또한 vimentin, PLK1, E-cadherin과 N-cadherin의 단백질량도 mRNA 발현량과 같은 결과를 관찰한 결과 같은 양상을 보여주었으며, 특히 PLK1의 활성형인 T210 잔기에서의 인산화 단백질 양이 대조군에 비해 TGF-β 처리군에서 높게 증가되는 것을 관찰할 수 있었다(도 2D). 단백질 양의 상대적 변화를 그래프로 표시하였다 (도 2D, 오른쪽 그림).To this end, to observe changes in the expression of PLK1 and FoxM1 and activation of PLK1 in the process of inducing epithelial-mesenchymal transition (EMT), a cancer metastasis cell model, by treating TGF-β, non-small cell lung cancer cell lines A549, NCI-H358, and NCI -H460 cell lines were treated with TGF-β to induce epithelial-mesenchymal transition, and then the mRNA and protein expression levels were analyzed. First, in the group treated with TGF-β, an increase in the mRNA expression level of the mesenchymal marker CDH2, SNAl1 and SNAI2 and a decrease in the mRNA expression level of the epithelial marker CDH1 were observed. Under these conditions, an increase in the mRNA expression levels of PLK1 and FOXM1 was observed compared to the control group (Figures 2A, 2B, 2C). In addition, the protein amounts of vimentin, PLK1, E-cadherin, and N-cadherin were observed to show the same results as the mRNA expression levels, and showed the same pattern. In particular, the amount of phosphorylated protein at the T210 residue, the active form of PLK1, was higher than that of TGF-β compared to the control group. A high increase was observed in the treatment group (Figure 2D). Relative changes in protein amounts were displayed graphically (Figure 2D, right figure).
PLK1과 FoxM1과의 상관성 분석과 FoxM1의 인산화가 EMT에 의존하는지 여부를 관찰하기 위해서 A549 및 NCI-H460 세포에 TGF-β를 처리한 세포에 탈인산화효소(Phosphatase, CIP)를 처리하여 p-FoxM1, p-PLK1, p-TCTP의 인산화 정도를 겔 지연과 인산화항체를 활용하여 면역침강법과 면역블랏법을 이용하여 분석하였다. 연구결과, 탈인산화효소 처리로 TGF-β 처리에 의해 상향 이동된 PLK1, TCTP 및 FoxM1의 밴드의 이동성을 지연시킴을 알 수 있었다. 또한, 인산화항체를 활용하여 검토한 결과, p-FoxM1 Ser, p-PLK1 T210 및 p-TCTP S46의 수준이 탈인산화효소 처리에 의해 감소됨을 확인하였다(도 2E, 2F). 이는 TGF-β 처리로 유도된 EMT동안 FOXM1과 PLK1이 인산화됨을 알 수 있었다. 따라서 FoxM1과 PLK1은 비소세포폐암 전이가 유도되는 환경에서 그 발현이 높으며 PLK1 활성 증가 및 FoxM1의 인산화와 비례 관계가 있어 암전이에서 이들 인자의 인산화와의 상관관계가 있음을 알 수 있었다.To analyze the correlation between PLK1 and FoxM1 and to observe whether phosphorylation of FoxM1 depends on EMT, A549 and NCI-H460 cells treated with TGF-β were treated with phosphatase (CIP) to produce p-FoxM1. , the degree of phosphorylation of p-PLK1 and p-TCTP was analyzed using immunoprecipitation and immunoblotting using gel retardation and phosphorylated antibodies. As a result of the study, it was found that dephosphorylation enzyme treatment delayed the mobility of the bands of PLK1, TCTP, and FoxM1, which were upwardly moved by TGF-β treatment. In addition, as a result of examination using a phosphorylation antibody, it was confirmed that the levels of p-FoxM1 Ser, p-PLK1 T210, and p-TCTP S46 were reduced by dephosphorylation enzyme treatment (Figures 2E, 2F). This showed that FOXM1 and PLK1 were phosphorylated during EMT induced by TGF-β treatment. Therefore, the expression of FoxM1 and PLK1 is high in an environment where non-small cell lung cancer metastasis is induced, and there is a proportional relationship with increased PLK1 activity and phosphorylation of FoxM1, indicating a correlation with phosphorylation of these factors in cancer metastasis.
본 발명의 발명자는 TGF-β에 의해 유도한 전이성 폐암세포에서 활성형 PLK1에 의한 FoxM1의 인산화 및 신규 인산화 부위를 확인하였다 (도 3)The inventor of the present invention identified phosphorylation of FoxM1 by activated PLK1 and a new phosphorylation site in metastatic lung cancer cells induced by TGF-β (Figure 3)
TGF-β처리에 의해서 유도된 암전이 조건에서 PLK1과 FoxM1의 상호작용을 탐구하기 위하여 면역침강법을 수행하였다. 먼저 Myc-tagged FoxM1을 발현시키고 TGF-β를 처리한 후 얻은 세포 용해액을 이용하여, 아가로오스 비드와 PLK1 항체로 PLK1 단백질을 침강하였으며 면역블롯법을 통해서 상호작용하는 단백질을 분석하였다. 연구결과, Myc-tagged FoxM1을 발현시켜 TGF-β를 처리한 조건에서 FoxM1과 PLK1이 서로 결합함을 확인하였다(도 3A). 또한 비소세포폐암 내부에 존재하는 내인성 FoxM1이 TGF-β처리에 의해 PLK1과 결합할 수 있는 지 A549 세포(도 3B)와 NCI-H460 세포(도 3C)에서 관찰한 결과, TGF-β에 의해 암전이를 유도시킨 실험군에서 PLK1과 FoxM1의 결합이 증가함을 관찰하였다(도 3B, 3C). 따라서 TGF-β에 의해 유도된 암전이 과정에서 PLK1과 FoxM의 상호작용이 증가함을 관찰할 수 있었다. Immunoprecipitation was performed to explore the interaction between PLK1 and FoxM1 in cancer metastasis conditions induced by TGF-β treatment. First, using the cell lysate obtained after expressing Myc-tagged FoxM1 and treating with TGF-β, PLK1 protein was precipitated with agarose beads and PLK1 antibody, and interacting proteins were analyzed through immunoblotting. As a result of the study, it was confirmed that FoxM1 and PLK1 bind to each other under conditions in which Myc-tagged FoxM1 was expressed and treated with TGF-β (Figure 3A). In addition, we observed in A549 cells (Figure 3B) and NCI-H460 cells (Figure 3C) whether endogenous FoxM1, which exists inside non-small cell lung cancer, can bind to PLK1 by TGF-β treatment. As a result, cancer metastasis by TGF-β was observed. In the experimental group that induced this, an increase in the binding of PLK1 and FoxM1 was observed (Figures 3B, 3C). Therefore, it was observed that the interaction between PLK1 and FoxM increased during the cancer metastasis process induced by TGF-β.
FoxM1의 인산화는 세포주기에서 세포분열기에 필요로 하는 다양한 인자들의 발현을 조절하는 전사인자로 알려져 있다. PLK1에 의한 FoxM1의 715번과 729번 위치의 세린(S) 부위에 인산화가 보고되어 있는데 이 부위에서의 인산화는 세포분열을 촉진시키는 기능으로 알려져 있다 (FU, Zheng et al., Nature cell biology (2008) 10.9:1076-1082). 활성화된 PLK1에 의한 전이성이 증가되는 조건에서 이러한 부위의 점 돌연변이체들이 암세포의 전이성이나 침습성, 종양형성을 차단하는 지에 대한 연구내용은 보고되지 않았다. PLK1은 EMT과정에서 활성화되는 종양인산화효소 단백질로(Shin et al., Oncogene (2020) 39(4) 767-785) 두 단백질의 상호작용으로 FoxM1이 PLK1에 의해 인산화되는지 확인하고자 인산화효소 반응법을 수행하였다. Phosphorylation of FoxM1 is known to be a transcription factor that regulates the expression of various factors required for cell division in the cell cycle. Phosphorylation at the serine (S) site at positions 715 and 729 of FoxM1 by PLK1 has been reported, and phosphorylation at this site is known to promote cell division (FU, Zheng et al., Nature cell biology (FU) 2008) 10.9:1076-1082). No studies have been reported on whether point mutations in these regions block the metastasis, invasiveness, or tumor formation of cancer cells under conditions where metastatic properties are increased by activated PLK1. PLK1 is a tumor phosphatase protein that is activated during the EMT process (Shin et al., Oncogene (2020) 39(4) 767-785). To determine whether FoxM1 is phosphorylated by PLK1 due to the interaction of the two proteins, a phosphatase reaction method was used. carried out.
본 발명에서는 PLK1에 FoxM1의 인산화 부위를 찾기 위하여 액체크로마토그래피 질량분석법과 인산화효소 반응법을 진행하였다. 세린/트레오닌 인산화효소인 PLK1이 FoxM1을 기질로서 인산화시키는지 증명하고자 정제된 FoxM1 원형과 활성형 PLK1-T210D을 방사선이 표지된 r32-P-ATP을 함께 넣고 효소반응을 진행하였다. FoxM1은 PLK1의 기질로 알려진 양성 대조군인 TCTP과 유사한 수준으로 강하게 활성형 PLK1-TD에 의해 인산화되었다(도 3D). 또한 PLK1에 의한 FoxM1의 인산화 부위를 찾기 위하여 인산화 반응 후 액체크로마토그래피 질량분석법을 진행하였다. 본 분석법으로 FoxM1의 Ser-25, Ser-360, Ser-361, Ser-393가 PLK1에 의해 인산화되는 부분으로 예측되었다(도 3E). 예측된 FoxM1의 인산화 부분 4곳과 PLK1에 의한 FoxM1을 세포주기동안 인산화시키는 것으로 기 보고된 Ser-715 잔기를 부분 특이적 돌연변이 치환법(Site-specific mutagenesis)을 이용하여 알라닌으로 치환시켜 탈인산화 돌연변이체로 제작하였다. 만들어진 돌연변이체를 GST가 표지된 단백질로 분리 정제하여 생산하였고 이를 동일한 인산화효소 반응법을 수행하였다. FoxM1의 인산화 예측 부위 5가지 중에서 Ser-25, Ser-361, Ser715의 알라닌 돌연변이체가 원형과 비교했을 때 인산화 정도가 두드러지게 감소하였다(도 3F). 이러한 연구결과로, PLK1이 FoxM1의 Ser-25, Ser-361, Ser-715 잔기를 인산화시킨다는 것을 알 수 있었다. In the present invention, liquid chromatography mass spectrometry and phosphorylase reaction method were performed to find the phosphorylation site of FoxM1 in PLK1. To prove that PLK1, a serine/threonine phosphatase, phosphorylates FoxM1 as a substrate, purified original FoxM1 and activated PLK1-T210D were added together with radiolabeled r32-P-ATP and an enzymatic reaction was performed. FoxM1 was strongly phosphorylated by active PLK1-TD at a level similar to that of TCTP, a positive control known to be a substrate of PLK1 (Figure 3D). In addition, to find the phosphorylation site of FoxM1 by PLK1, liquid chromatography mass spectrometry was performed after the phosphorylation reaction. Using this analysis, Ser-25, Ser-360, Ser-361, and Ser-393 of FoxM1 were predicted to be phosphorylated by PLK1 (Figure 3E). Dephosphorylation mutation was achieved by substituting the four predicted phosphorylation sites of FoxM1 and the Ser-715 residue, previously reported to phosphorylate FoxM1 by PLK1 during the cell cycle, with alanine using site-specific mutagenesis. Made with a sieve. The resulting mutant was purified and produced as a GST-labeled protein, which was then subjected to the same phosphatase reaction method. Among the five predicted phosphorylation sites of FoxM1, the degree of phosphorylation of the alanine mutants at Ser-25, Ser-361, and Ser715 was significantly reduced compared to the original (Figure 3F). As a result of this study, it was found that PLK1 phosphorylates the Ser-25, Ser-361, and Ser-715 residues of FoxM1.
또한, 본 발명은 PLK1에 의한 FoxM1의 신규 인산화부위에서의 인산화 점 돌연변이체 단백질에 의한 암세포의 전이성 촉진 효과 및 FoxM1의 비인산화 점 돌연변이체 단백질에 의한 이에 대한 억제 효과를 제공한다 (도 4). In addition, the present invention provides an effect of promoting metastasis of cancer cells by a phosphorylated point mutant protein at a new phosphorylation site of FoxM1 by PLK1 and an inhibitory effect thereof by a non-phosphorylated point mutant protein of FoxM1 (FIG. 4).
PLK1에 의한 FoxM1의 인산화와 비인산화 점 돌연변이체를 발현하는 단백질이 암세포의 침습성과 이동성에 관여하는 지 알아보고자 독시사이클린(doxycycline) 처리에 의해 발현유도조절이 가능한 렌티바이러스 시스템을 이용하여 안정한 세포주를 구축한 후에 각 변이체를 발현시켜 각 변이체의 암세포의 침습성과 이동성에 미치는 효과를 관찰하였다. 여러 인산화변이체 중에서 Ser25에 대한 인산화 점 돌연변이체인 S25E 발현 세포에서 EMT 마커 중 N-cadherin (CDH2)의 발현이 증가됨을 qRT-PCR과 웨스턴 블롯에서 관찰할 수 있었다. 이러한 효과는 Ser25에 대한 비인산화 점 돌연변이체인 S25A 발현되는 세포군에서 감소되어 억제됨을 알 수 있었다(도 4A, 4B). 각 돌연변이체의 발현이 세포 증식(cell proliferation)에 미치는 효과를 관찰하고자 세포 증식 검정법을 진행하여 모든 돌연변이 형태에서 관찰한 결과 S715E에서 세포 증식이 현저히 증가함을 관찰할 수 있었는데, 이는 TGF-β처리한 세포군 보다 더 증가함을 알 수 있었다. 또한 세포이동성이 증가되었던 인산화 점 돌연변이체 S25E 발현 세포의 경우 세포증식이 현저히 증가하지는 않음을 관찰하였다(도 4C).To investigate whether proteins expressing phosphorylation and non-phosphorylation point mutants of FoxM1 by PLK1 are involved in the invasiveness and mobility of cancer cells, a stable cell line was constructed using a lentivirus system capable of inducing and controlling expression by doxycycline treatment. Afterwards, each variant was expressed and the effects of each variant on the invasiveness and mobility of cancer cells were observed. Among several phosphorylation variants, qRT-PCR and Western blot showed that the expression of N-cadherin (CDH2), an EMT marker, was increased in cells expressing S25E, a phosphorylation point mutant for Ser25. This effect was found to be reduced and suppressed in the cell population expressing S25A, a non-phosphorylated point mutant for Ser25 (Figures 4A, 4B). A cell proliferation assay was performed to observe the effect of the expression of each mutant on cell proliferation. As a result of observing all mutant forms, it was observed that cell proliferation was significantly increased in S715E, which was observed after TGF-β treatment. It was found that it increased more than one cell group. Additionally, in the case of cells expressing the phosphorylated point mutant S25E, which had increased cell mobility, it was observed that cell proliferation did not significantly increase (Figure 4C).
FoxM1의 인산화 및 비인산화 점 돌연변이체 각 단백질이 발현되는 A549 세포에서 암세포의 전이성을 관찰하기 위하여 Transwell을 활용한 암세포 이동성 실험(migration assay)을 진행하였다(도 4D). 연구결과 FoxM1의 S25 잔기의 인산화 점 돌연변이체(S25E)가 발현되는 세포군에서는 대조군 대비 6배가량 증가되었는데, 이는 양성 대조군인 TGF-β 처리 세포군(5ng/ml)에서 4배가량 증가된 것과 비교해도 더 많은 암세포가 이동함을 알 수 있었다. 이와 반대로 비인산화 점 돌연변이체(S25A) 단백질을 발현하는 암세포의 이동성은 감소되었다. 하지만 FoxM1의 S361 잔기와 S715 잔기 변이체를 발현시킨 세포군에서는 인산화 및 비인산화 점 돌연변이체 사이에 이동성에서 별다른 차이가 없음을 관찰하였다. 따라서 FoxM1 S25 잔기의 인산화 점 돌연변이체(S25E)가 발현되는 세포군에서 암세포의 이동성이 증가되며 반면에 비인산화 점 돌연변이체인 S25A 발현되는 세포에서는 현저히 감소됨을 관찰할 수 있었다. To observe metastatic properties of cancer cells in A549 cells expressing each phosphorylated and non-phosphorylated point mutant protein of FoxM1, a cancer cell migration assay was performed using Transwell (Figure 4D). As a result of the study, in the cell group expressing the phosphorylated point mutant (S25E) of the S25 residue of FoxM1, there was an approximately 6-fold increase compared to the control group, which was compared to an approximately 4-fold increase in the TGF-β treated cell group (5 ng/ml), which was a positive control. It was found that more cancer cells were moving. In contrast, the mobility of cancer cells expressing the non-phosphorylated point mutant (S25A) protein was reduced. However, in the cell group expressing the S361 residue and S715 residue variants of FoxM1, no significant difference in mobility was observed between the phosphorylated and non-phosphorylated point mutants. Therefore, it was observed that the mobility of cancer cells was increased in the cell group expressing the phosphorylated point mutant (S25E) of the FoxM1 S25 residue, while it was significantly decreased in the cells expressing S25A, a non-phosphorylated point mutant.
본 발명의 발명자는 FoxM1의 인산화 점 돌연변이체에 의한 암세포에서의 침습성 촉진 효과 및 FoxM1의 비인산화 점 돌연변이체에 의한 암세포의 침습성 억제 효과를 확인하였다.The inventors of the present invention confirmed the invasiveness-promoting effect of cancer cells by a phosphorylated point mutant of FoxM1 and the invasiveness-inhibiting effect of cancer cells by a non-phosphorylated point mutant of FoxM1.
FoxM1의 인산화 점 돌연변이체와 비인산화 점 돌연변이체을 이용하여 암세포에서의 침습성 촉진 및 억제 효과에 대한 실험을 진행하였다. 마트리겔을 이용한 침습성 분석(invasion assay)를 이용하여 암세포의 침습성을 관찰하고자 하였다(도 4E). 먼저, 침습능을 평가하기 위하여 마트리겔 인서트에 각각의 FoxM1의 점 돌연변이체를 발현하는 세포를 혈청이 없는 배지와 함께 분주하고 실험플레이트에 혈청이 포함된 배지를 분주하여 5일 동안 배양하였다. 침습된 암세포를 크리스탈 바이올렛으로 염색하여 관찰하고 DMSO로 녹인 후 590nm의 파장에서 흡광도를 측정하였다. 연구결과, FoxM1의 인산화 점 돌연변이체 단백질을 발현하는 폐암세포군에서 대조군과 FoxM1의 원형 대비 침습성이 증가됨을 관찰하였다. 특히 FoxM1의 S25E 인산화 점 돌연변이체에서 가장 높은 암세포의 침습성을 관찰할 수 있었다. 반면에 FoxM1의 비인산화 점 돌연변이체 단백질을 발현하는 폐암세포군에서는 침습성이 감소됨을 관찰하였다. 따라서 폐암세포에서 FoxM1의 S25 잔기에서의 인산화 점 돌연변이체에 의한 암세포에서의 침습성 촉진 효과 및 FoxM1의 비인산화 점 돌연변이체에 의한 암세포의 침습성 억제 효과를 관찰할 수 있었다.Experiments were conducted on the promoting and suppressing effects of invasiveness in cancer cells using phosphorylated and non-phosphorylated point mutants of FoxM1. We attempted to observe the invasiveness of cancer cells using an invasion assay using Matrigel (Figure 4E). First, to evaluate the invasion ability, cells expressing each point mutant of FoxM1 were seeded on the Matrigel insert with serum-free medium, and serum-containing medium was distributed on the experimental plate and cultured for 5 days. Invaded cancer cells were observed by staining with crystal violet, dissolved in DMSO, and absorbance was measured at a wavelength of 590 nm. As a result of the study, it was observed that the invasiveness of the lung cancer cell group expressing the phosphorylated point mutant protein of FoxM1 was increased compared to the control group and the original FoxM1 protein. In particular, the highest cancer cell invasiveness was observed in the S25E phosphorylation point mutant of FoxM1. On the other hand, a decrease in invasiveness was observed in the lung cancer cell group expressing a non-phosphorylated point mutant protein of FoxM1. Therefore, in lung cancer cells, the invasiveness-promoting effect in cancer cells by a phosphorylated point mutant at the S25 residue of FoxM1 and the invasiveness-inhibiting effect of cancer cells by a non-phosphorylated point mutant of FoxM1 were observed.
그리고 FoxM1의 각 인산화 점 돌연변이체(S25E, S361E, S715E)와 3개 점 동시 돌연변이체(EEE) 단백질이 발현되는 A549 세포에서 암세포의 이동성을 관찰하기 위해 상처 치유 분석 실험(Wound healing assay)을 진행하였다(도 4F, 4G). 각각 0h, 24h, 48h, 72h에서 현미경으로 세포간의 치유 각격을 측정함으로 하여 세포의 이동성을 관찰하였다(도 4F). 그리고 72시간에서 대조군을 0으로 하여 상대적 거리를 막대 그라프로 나타냈다(도 4G). 연구결과 FoxM1의 S25 잔기의 인산화 점 돌연변이체(S25E)가 발현되는 세포군에서는 대조군 대비 이동성이 증가되었는데, 이는 양성 대조군인 TGF-β 처리 세포군(5ng/ml)과 비슷하게 증가된 것을 알 수 있었다. 인산화 점 돌연변이체(S361E)와 인산화 점 돌연변이체(S717E) 단백질을 발현하는 암세포의 이동성은 대조군과 대비 별다른 차이가 없음을 관찰하였다. 또한, 3개 점 동시 돌연변이체(EEE)는 점 돌연변이체(S25E)에 비해 이동성이 약하지만 대조군 대비 증가한 것을 관찰할 수 있었다. 따라서 FoxM1 S25 잔기의 인산화 점 돌연변이체(S25E)가 발현되는 세포군에서 암세포의 이동성이 증가되며 다른 두 개 인산화 점 돌연변이체 S361E와 S715E 단백질 발현되는 세포에서는 이동성이 크게 증가하지 않음을 알 수 있었다.In addition, a wound healing assay was performed to observe the mobility of cancer cells in A549 cells expressing each phosphorylated point mutant of FoxM1 (S25E, S361E, S715E) and three point simultaneous mutant (EEE) proteins. (Figures 4F, 4G). Cell mobility was observed by measuring the healing gap between cells under a microscope at 0 h, 24 h, 48 h, and 72 h, respectively (Figure 4F). And at 72 hours, the relative distance was displayed as a bar graph, with the control group set as 0 (Figure 4G). As a result of the study, the mobility of the cell group expressing the phosphorylated point mutant (S25E) of the S25 residue of FoxM1 was found to be increased compared to the control group, which was similar to the positive control group of TGF-β treated cells (5 ng/ml). It was observed that the mobility of cancer cells expressing the phosphorylated point mutant (S361E) and phosphorylated point mutant (S717E) proteins showed no significant difference compared to the control group. In addition, the three point simultaneous mutant (EEE) had weaker mobility than the point mutant (S25E), but increased mobility was observed compared to the control group. Therefore, it was found that the mobility of cancer cells was increased in the cell group expressing the phosphorylated point mutant (S25E) of the FoxM1 S25 residue, while the mobility was not significantly increased in the cells expressing the other two phosphorylated point mutant S361E and S715E proteins.
또한, 본 발명은 PLK1에 의한 FoxM1의 신규 인산화부위인 S25 잔기에서의 인산화 점 돌연변이체 단백질에 의한 암세포의 원발소암으로서의 종양 형성능과 전이성 촉진 효과 및 FoxM1의 비인산화 점 돌연변이체 단백질에 의한 이에 대한 억제 효과를 제공한다. (도 5) In addition, the present invention promotes the tumorigenicity and metastasis of cancer cells as primary cancer by phosphorylated point mutant protein at S25 residue, a new phosphorylation site of FoxM1 by PLK1, and its inhibition by non-phosphorylated point mutant protein of FoxM1. Provides effect. (Figure 5)
마우스를 이용한 꼬리 정맥 주사 전이암 동물모델에서 PLK1에 의한 FoxM1의 인산화와 비인산화 점 돌연변이체 단백질을 발현하는 세포가 암전이와 종양형성에 관여하는 지 알아보고자 BALB/c nude 마우스에 FoxM1 S25E와 S25A 돌연변이체를 발현하는 A549 세포를 꼬리 정맥 주사를 하였다. 12주 동안 사육한 후 개복하여 장기에 암세포의 전이 및 종양형성 정도를 관찰하였다(도 5A). FoxM1의 S25E 인산화 점 돌연변이체를 발현하는 세포를 주입한 동물군의 폐에서 대조군, 원형, 그리고 비인산화 점 돌연변이체를 발현하는 동물군에 비해 폐장기로의 암전이 및 종양 형성이 현저히 높음을 관찰하였다. 반면, FoxM1의 비인산화 점 돌연변이체를 발현하는 세포를 주입한 동물군의 폐에서는 종양이 형성되지 않음을 확인할 수 있었다. 따라서 FoxM1의 PLK1에 의한 신규 인산화부위에서의 인산화 점 돌연변이체 단백질 발현에 의한 암세포의 종양형성을 촉진하는 효과를 관찰할 수 있었으며, FoxM1의 비인산화 점 돌연변이체 단백질에 의한 암세포의 종양 형성능 억제 효과를 관찰하였다.To investigate whether phosphorylation of FoxM1 by PLK1 and cells expressing non-phosphorylated point mutant proteins are involved in cancer metastasis and tumor formation in a metastatic cancer animal model by tail vein injection using mice, BALB/c nude mice were administered FoxM1 S25E and S25A. A549 cells expressing the mutants were injected through the tail vein. After rearing for 12 weeks, the animals were laparotomized to observe the degree of metastasis and tumor formation of cancer cells in the organs (Figure 5A). It was observed that cancer metastasis to lung organs and tumor formation were significantly higher in the lungs of animal groups injected with cells expressing the S25E phosphorylated point mutant of FoxM1 compared to the control, prototype, and animal groups expressing the non-phosphorylated point mutant. . On the other hand, it was confirmed that no tumors were formed in the lungs of animals injected with cells expressing a non-phosphorylated point mutant of FoxM1. Therefore, the effect of promoting tumorigenesis of cancer cells was observed by the expression of phosphorylated point mutant protein at the new phosphorylation site by PLK1 of FoxM1, and the effect of suppressing the tumorigenicity of cancer cells by non-phosphorylated point mutant protein of FoxM1 was observed. observed.
H&E(Haematoxylin and eosin)와 Ki67 염색을 통해서 암세포 증식정도를 관찰하고자 하였다. 연구 결과, FoxM1 인산화 점 돌연변이체 실험군에서 암세포 증식정도가 높으며 비인산화 점 돌연변이체 실험군에서는 현저히 낮은 것을 관찰할 수 있었다(도 5B 및 5C). 이는 동물모델에서 FoxM1의 인산화 점 돌연변이체를 발현하는 암세포가 암전이성과 종양형성능을 촉진시키며, 반대로 비인산화 점 돌연변이체는 암전이성과 종양형성능을 억제하는 것을 확인할 수 있었다.We attempted to observe the degree of cancer cell proliferation through H&E (Haematoxylin and eosin) and Ki67 staining. As a result of the study, it was observed that the degree of cancer cell proliferation was high in the FoxM1 phosphorylated point mutant experimental group and significantly low in the non-phosphorylated point mutant experimental group (Figures 5B and 5C). In an animal model, it was confirmed that cancer cells expressing a phosphorylated point mutant of FoxM1 promoted cancer metastasis and tumorigenicity, while non-phosphorylated point mutants suppressed cancer metastasis and tumorigenic ability.
다음으로 폐 조직의 일부를 용해하여 상피간엽이행 마커의 단백질 수준을 관찰한 결과, FoxM1 인산화 점 돌연변이체의 실험군에서 중간엽 마커인 N-cadherin의 단백질 수준이 증가하였으며, E-cadherin의 수준은 감소하였다. 또한 면역회피에 관여하여 암세포의 종양형성능을 증가시킨다고 알려진 PD-L1의 발현도 각 실험군에서 확인한 결과, FoxM1 인산화 점 돌연변이체 실험군에서 발현이 높게 관찰되었다(도 5D). 추가로 단백질 수준뿐만 아니라 mRNA 수준에서도 상위와 같은 결과를 확인하였다(도 5E). 따라서 FoxM1의 S25에서 PLK1에 의한 인산화는 상피간엽이행과 전이성과 종양형성능을 증진시킴을 시사한다. 반면 비인산화 점 돌연변이체는 상피간엽이행과 종양형성능을 억제하는 효과가 뛰어남을 알 수 있다. Next, a portion of the lung tissue was lysed and the protein level of the epithelial-mesenchymal transition marker was observed. As a result, the protein level of N-cadherin, a mesenchymal marker, increased, and the level of E-cadherin decreased in the experimental group of FoxM1 phosphorylation point mutants. did. In addition, the expression of PD-L1, which is known to be involved in immune evasion and increase the tumorigenic ability of cancer cells, was checked in each experimental group. As a result, high expression was observed in the FoxM1 phosphorylation point mutant experimental group (Figure 5D). Additionally, similar results were confirmed not only at the protein level but also at the mRNA level (Figure 5E). Therefore, it suggests that phosphorylation by PLK1 at S25 of FoxM1 promotes epithelial-mesenchymal transition, metastasis, and tumorigenic potential. On the other hand, it can be seen that the non-phosphorylated point mutant has an excellent effect in suppressing epithelial-mesenchymal transition and tumorigenic ability.
FoxM1의 인산화 및 비인산화 점 돌연변이체 각 단백질이 발현되는 A549 세포에서 암세포의 세포사멸 정도를 관찰하기 위하여 caspase-3 효소의 활성을 측정하였다(도 5F). FoxM1 비인산화 점 돌연변이체가 과발현된 세포에서 caspase-3 효소의 활성이 가장 높게 측정된 것으로 나타났으며, 이를 통해 FoxM1 비인산화 점 돌연변이체의 과발현은 세포사멸을 유도 및 증가시킴을 알 수 있었다.To observe the degree of cancer cell apoptosis in A549 cells expressing each phosphorylated and non-phosphorylated point mutant protein of FoxM1, caspase-3 enzyme activity was measured (Figure 5F). The caspase-3 enzyme activity was found to be highest in cells in which the FoxM1 non-phosphorylated point mutant was overexpressed. This showed that overexpression of the FoxM1 non-phosphorylated point mutant induced and increased apoptosis.
또한, 본 발명은 FoxM1의 인산화 점 돌연변이체에 의한 단핵구의 암세포로의 recruitment 촉진 효과 및 단핵구의 종양과 관련된 대식세포로의 분화를 FoxM1의 비인산화 점 돌연변이체에 의해 억제하는 효과를 제공한다 (도 6). In addition, the present invention provides the effect of promoting the recruitment of monocytes to cancer cells by a phosphorylated point mutant of FoxM1 and the effect of suppressing the differentiation of monocytes into tumor-related macrophages by a non-phosphorylated point mutant of FoxM1 (Figure 6).
FoxM1은 대식세포의 이동 및 분화 등에 관여한다는 연구결과(BALLI, David et al., Oncogene (2012) 31.34:3875-3888; YANG, Yang et al., Diabetes Research and Clinical Practice (2022) 184: 109121)를 기반으로 FoxM1 S25E가 발현되는 암세포가 대식세포 (Macrophage cell)의 분화(polarization)에 영향을 미치는지 관찰하고자 인간 대식세포인 THP-1 세포와 FoxM1 점 돌연변이를 과발현한는 A549 세포를 48시간동안 공배양 후에 M1, M2 마커를 관찰하였다. 그 결과 M1 마커인인 INOS, IL12B의 발현은 변화가 없으나 M2 마커인인 IL10, CD163, CD206, TGFB1, VEGFA 가 모두 FoxM1 인산화 점 돌연변이체(S25E)가 과발현된 세포에서 증가하였다(도 6A). 그중 CD206, TGFB1, VEGFA는 M2d-종양 관련 대식세포(M2d-TAM)의 마커(JAYASINGAM et al., Front. Oncol (2020) 9:1512)로 알려져 있어서 FoxM1 점 인산화 돌연변이체(S25E)가 발현되는 A549 세포와 공배양한 THP-1 세포에서 이들 인자의 상향 조절은 A549 세포가 THP-1 세포를 M2d-TAM로 분화시킴을 알 수 있었다. Research results show that FoxM1 is involved in the migration and differentiation of macrophages (BALLI, David et al., Oncogene (2012) 31.34:3875-3888; YANG, Yang et al., Diabetes Research and Clinical Practice (2022) 184: 109121) Based on this, to observe whether cancer cells expressing FoxM1 S25E affect polarization of macrophages, THP-1 cells, which are human macrophages, and A549 cells overexpressing FoxM1 point mutations were co-cultured for 48 hours. Afterwards, M1 and M2 markers were observed. As a result, there was no change in the expression of the M1 markers INOS and IL12B, but the M2 markers IL10, CD163, CD206, TGFB1, and VEGFA were all increased in cells overexpressing the FoxM1 phosphorylation point mutant (S25E) (Figure 6A). Among them, CD206, TGFB1, and VEGFA are known to be markers of M2d-tumor-associated macrophages (M2d-TAM) (JAYASINGAM et al., Front. Oncol (2020) 9:1512), so they are characterized by expression of the FoxM1 point phosphorylation mutant (S25E). Upregulation of these factors in THP-1 cells co-cultured with A549 cells indicated that A549 cells differentiated THP-1 cells into M2d-TAM.
또한 FoxM1 점 인산화 돌연변이(S25E)가 발현되는 A549 세포에서 M2d 유도 인자인 IL4, IL6, IL10, VEGFA를 발현수준을 분석한 결과, 이들 인자들의 발현이 증가되었으며 FoxM1 비인산화 변이체(S25A)가 발현되는 A549 세포에서는 현저히 감소됨을 관찰하였다(도 6B). 이러한 현상은 FoxM1 변이체가 발현되는 A549 세포 배양 배지에서 TGFB1, VEGFA 단백질의 양을 ELISA로 측정한 결과, S25E에서는 현저히 증가되며 S25A에서는 강하게 감소됨을 관찰하였다(도 6C). In addition, as a result of analyzing the expression levels of M2d-inducing factors IL4, IL6, IL10, and VEGFA in A549 cells expressing FoxM1 point phosphorylation mutant (S25E), the expression of these factors was increased, and the expression of these factors was increased in A549 cells expressing FoxM1 point phosphorylation mutant (S25E). A significant decrease was observed in A549 cells (Figure 6B). This phenomenon was observed to be significantly increased in S25E and strongly decreased in S25A, as a result of measuring the amount of TGFB1 and VEGFA proteins in the A549 cell culture medium expressing the FoxM1 variant by ELISA (Figure 6C).
다음으로는 FoxM1 점 인산화 돌연변이체(S25E)에 의해 M2 대식세포로의 분화에 직접적으로 영향을 미치는 지를 관찰하고자 FoxM1의 발현을 억제하는 발현억제 시스템을 구축하였다. FoxM1를 발현 억제한 세포와 THP-1세포와 공배양한 후 THP-1 세포에서 M2 marker인 CD163, CD206, VEGFA의 발현이 현저히 감소됨을 관찰하였다. 또한 FoxM1 점 인산화 돌연변이체(S25E)를 발현시킨 암세포와 THP-1 세포를 공배양한 후에 THP-1 세포에서 대조군(Mock_shCtrl) 대비 M2 marker인 CD163, CD206, VEGFA의 발현이 다시 증가함을 확인하였다(도 6D). 즉, M2 대식세포로의 분화에 FoxM1 인산화 점 돌연변이체(S25E)가 직접적으로 영향을 미침을 의미한다. Next, we constructed an expression suppression system to suppress the expression of FoxM1 to observe whether the FoxM1 point phosphorylation mutant (S25E) directly affects differentiation into M2 macrophages. After co-culturing cells with suppressed expression of FoxM1 and THP-1 cells, it was observed that the expression of M2 markers CD163, CD206, and VEGFA in THP-1 cells was significantly reduced. In addition, after co-culturing THP-1 cells with cancer cells expressing the FoxM1 point phosphorylation mutant (S25E), it was confirmed that the expression of M2 markers CD163, CD206, and VEGFA increased again in THP-1 cells compared to the control group (Mock_shCtrl). (Figure 6D). In other words, this means that the FoxM1 phosphorylation point mutant (S25E) directly affects differentiation into M2 macrophages.
또한 마우스의 폐조직에서 광범위 대식세포 마커인 CD68와 종양관련대식세포 마커인 CD163 마커를 면역염색법으로 분석한 결과, FoxM1 인산화 점 돌연변이체(S25E)가 과발현된 A549 세포를 주사한 마우스 폐조직에서 CD68, CD163 마커의 발현이 높게 관찰됨을 확인하였다(도 6E, 6F). 반면, 비인산화 점 돌연변이체(S25A)를 과발현시킨 A549 세포를 주사한 폐조직에서는 CD68, CD163 마커의 발현이 현저히 낮음을 관찰하였다. 추가적으로 폐조직 용해액을 이용하여 면역블랏법으로 종양주변의 대식세포가 증가함을 관찰하고자 CD68, CD163 단백질의 발현을 관찰한 결과, FoxM1 인산화 점 돌연변이체(S25E)가 과발현된 A549 세포를 주사한 마우스 폐조직에서 이들 종양 대식세포 마커의 발현이 높은 반면, 비인산화 점 돌연변이체(S25A)를 과발현시킨 A549 세포를 주사한 마우스 폐조직에서 이들 종양 대식세포 마커의 발현이대조군보다 낮음을 관찰할 수 있었다(도 6G). 본 연구결과를 통하여 FoxM1 비인산화 점 돌연변이체가 종양관련 대식세포로의 분화를 억제하는 효과를 관찰할 수 있었다.In addition, as a result of immunostaining analysis of CD68, a broad macrophage marker, and CD163, a tumor-related macrophage marker, in mouse lung tissue, CD68 was detected in mouse lung tissue injected with A549 cells overexpressing the FoxM1 phosphorylated point mutant (S25E). , it was confirmed that high expression of the CD163 marker was observed (Figures 6E, 6F). On the other hand, in lung tissue injected with A549 cells overexpressing the non-phosphorylated point mutant (S25A), the expression of CD68 and CD163 markers was observed to be significantly low. In addition, the expression of CD68 and CD163 proteins was observed to observe the increase in macrophages around the tumor using lung tissue lysate by immunoblotting. As a result, A549 cells overexpressing FoxM1 phosphorylation point mutant (S25E) were injected. While the expression of these tumor macrophage markers was high in mouse lung tissue, the expression of these tumor macrophage markers in mouse lung tissue injected with A549 cells overexpressing the non-phosphorylated point mutant (S25A) was observed to be lower than that in the control group. There was (Figure 6G). Through the results of this study, we were able to observe the effect of FoxM1 non-phosphorylated point mutants on suppressing differentiation into tumor-related macrophages.
또한, 본 발명은 FoxM1의 인산화 점 돌연변이체에 의한 종양 면역회피반응 촉진 효과 및 FoxM1의 비인산화 점 돌연변이체에 의한 면역회피반응 억제 효과를 제공한다(도 7). In addition, the present invention provides an effect of promoting tumor immune evasion response by a phosphorylated point mutant of FoxM1 and an effect of suppressing immune evasion response by a non-phosphorylated point mutant of FoxM1 (FIG. 7).
종양관련 대식세포는 암세포의 면역회피를 도와주어 암세포의 생존을 증가시키는 것으로 보고되어 있다(NOY, Roy and POLLARD, Jeffrey W., Immunity (2014) 41.1: 49-61). 이를 기반으로 FoxM1 인산화 및 비인산화 점 돌연변이체가 발현되는 A549 세포와 단핵세포 THP-1 세포를 공배양하여 폐암세포의 생존율을 분석한 결과, A549 세포: THP-1세포의 비율이 1:0, 1:2, 1:4, 1:6로 변화함에 따라 FoxM1 S25E 인산화 점 돌연변이체를 발현하는 A549 (A549S25E)세포의 생존율이 점차 증가함으로 관찰하였다(도 7A). 반면, FoxM1 S비인산화 점 돌연변이체를 발현하는 A549 (A549S25A)세포의 생존율은 대조군과 비교하여 변화가 거의 없었다.Tumor-related macrophages are reported to increase the survival of cancer cells by helping them evade immunity (NOY, Roy and POLLARD, Jeffrey W., Immunity (2014) 41.1: 49-61). Based on this, the survival rate of lung cancer cells was analyzed by co-culturing A549 cells expressing FoxM1 phosphorylation and non-phosphorylation point mutants with mononuclear THP-1 cells, and the ratio of A549 cells: THP-1 cells was 1:0, 1:0. :2, 1:4, 1:6, the survival rate of A549 (A549 S25E ) cells expressing the FoxM1 S25E phosphorylation point mutant was observed to gradually increase (Figure 7A). On the other hand, the survival rate of A549 (A549S25A) cells expressing the FoxM1 S non-phosphorylated point mutant showed little change compared to the control group.
위의 각 세포에서 고형암 면역회피에 관여하는 PD-1과 PD-L1의 mRNA 발현수준을 검토한 결과, A549S25A세포와 공배양한 THP-1 단핵세포에서 면역회피 인자인 PD-1 mRNA (CD279) 발현이 제일 높은 것으로 관찰되었다(도 7B). 또한 인산화 점 돌연변이체 발현 (A549S25E) 세포에서 면역회피 인자 PD-L1의 mRNA (CD274)발현도 현저히 증가함을 관찰하였다. 흥미롭게도 비인산화 점 돌연변이체 발현 A549S25A 세포에서 면역회피 인자 PD-L1의 mRNA (CD274)발현이 대조군대비 50%정도 감소함을 관찰함으로써 FoxM1의 비인산화 점 돌연변이체는 암세포의 면역회피인자의 발현을 억제하는 효과를 보여주었다(도 7C).As a result of examining the mRNA expression levels of PD-1 and PD-L1, which are involved in immune evasion of solid tumors, in each of the above cells, PD-1 mRNA (CD279), an immune evasion factor, was found in THP-1 monocytes co-cultured with A549 S25A cells. ) expression was observed to be the highest (Figure 7B). In addition, it was observed that the mRNA (CD274) expression of the immune evasion factor PD-L1 (CD274) was significantly increased in cells expressing the phosphorylated point mutant (A549 S25E ). Interestingly, it was observed that the mRNA (CD274) expression of the immune evasion factor PD-L1 (CD274) in A549 S25A cells expressing the non-phosphorylated point mutant was reduced by about 50% compared to the control group, indicating that the non-phosphorylated point mutant of FoxM1 was associated with the expression of the immune evasion factor in cancer cells. showed an inhibitory effect (Figure 7C).
또한, T 세포인 Jurkat 세포과 FoxM1 인산화 및 비인산화 점 돌연변이체가 발현된 세포와 공배양을 통하여 T 세포의 종양면역능력에 미치는 효과를 관찰하고자 하였다(도 7D). Jurkat 세포과 공배양한 FoxM1 인산화 점 돌연변이체 발현 (A549S25E)세포의 생존율은 Jurkat 세포의 비율을 증가시킴에 따라 점차 증가되는 것이 관찰되었으나 A549S25A세포의 생존율은 원형이나 A549S25E 세포에 비해 현저히 억제됨을 관찰할 수 있었다(도 7D). 이는 A549S25E 세포가 T 세포의 성질에도 영향을 미칠 것으로 판단되므로, A549S25E 세포에 의한 tumor-infiltrating T lymphocytes (TILs)로의 분화를 검토하고자 공배양된 Jurkat 세포에서 TILs에서 발현되는 CD25와 CD29의 발현을 관찰하였다. 그 결과 A549S25E 세포와 공배양된 Jurkat 세포에서 CD25와 CD29의 발현이 현저히 증가됨을 관찰하였다. 하지만 A549S25A 세포와 공배양된 Jurkat 세포에서는 원형이나 인산화 점 돌연변이체를 발현하는 A549(A549S25E)세포와 공배양된 Jurkat 세포에 비해 현저히 억제하는 효과가 있음을 보여주었다(도 7E). 또한 A549S25E 세포에서 TILs 분화를 유도하는 IL6와 IL1A 및 종양면역회피인자인 CD274의 발현이 증가함을 관찰하였으나 A549S25A 세포에서 이들 인자의 발현은 현저히 낮음을 관찰하였다(도 7F). 추가적으로 종양미세환경에서 TAMs나 TILs에 의해 종양면역회피 반응에 A549S25A 세포가 억제효과를 있음을 THP-1세포 또는 Jurkat 세포와의 FoxM1 인산화 및 비인산화 점 돌연변이체가 과발현된 A549 세포와의 공배양 후에 생존율 변화 분석을 통하여 관찰하였다(도 7G). 따라서 본 발명은 FoxM1의 비인산화 점 돌연변이체에 의한 종양 면역회피반응 억제 효과를 제공한다. In addition, we attempted to observe the effect on the tumor immune capacity of T cells through co-culture with Jurkat cells, which are T cells, and cells expressing FoxM1 phosphorylated and non-phosphorylated point mutants (Figure 7D). The survival rate of FoxM1 phosphorylated point mutant-expressing (A549 S25E ) cells co-cultured with Jurkat cells was observed to gradually increase as the proportion of Jurkat cells increased, but the survival rate of A549 S25A cells was significantly suppressed compared to the original or A549 S25E cells. could be observed (Figure 7D). Since it is believed that A549 S25E cells may also affect the properties of T cells, the expression of CD25 and CD29 expressed on TILs in co-cultured Jurkat cells was conducted to examine the differentiation of A549 S25E cells into tumor-infiltrating T lymphocytes (TILs). was observed. As a result, it was observed that the expression of CD25 and CD29 was significantly increased in Jurkat cells co-cultured with A549 S25E cells. However, Jurkat cells co-cultured with A549 S25A cells showed a significant inhibitory effect compared to Jurkat cells co-cultured with A549 (A549 S25E ) cells expressing the original or phosphorylated point mutant (Figure 7E). In addition, we observed increased expression of IL6 and IL1A, which induce TIL differentiation, and CD274, a tumor immune evasion factor, in A549 S25E cells, but the expression of these factors was observed to be significantly low in A549 S25A cells (Figure 7F). Additionally, A549 S25A cells had an inhibitory effect on the tumor immune evasion response caused by TAMs or TILs in the tumor microenvironment after co-culture with A549 cells overexpressing FoxM1 phosphorylation and non-phosphorylation point mutants with THP-1 cells or Jurkat cells. Changes in survival rate were observed through analysis (Figure 7G). Therefore, the present invention provides the effect of suppressing tumor immune evasion response by a non-phosphorylated point mutant of FoxM1.
또한, 본 발명은 FoxM1의 비인산화 점 돌연변이 펩티드에 의한 암세포 침습성 및 이동성 및 종양 관련 대식세포(TAM)로의 분화 억제 효과를 제공한다(도 8). In addition, the present invention provides an inhibitory effect on cancer cell invasiveness and mobility and differentiation into tumor-associated macrophages (TAM) by a non-phosphorylated point mutant peptide of FoxM1 (FIG. 8).
단백질 및 핵산과 같은 거대 분자는 세포막을 투과할 수 없기 때문에 세포 내로 전달되기가 어렵다고 알려져 있다. 단백질을 세포 내로 전달하기 위해서 다양한 기술들이 개발되어 왔으며, 가장 대표적인 기술로는 세포 투과 펩티드(cell-penetrating peptide, CPP)가 있다. CPP는 8개에서 30개 사이의 아미노산으로 구성된 짧은 길이의 펩티드로 양이온성(cationic), 양친매성 (amphipathic), 혹은 소수성 (hydrophobic)의 특징을 갖는 것으로 나눈다. 세포막을 통과할 수 있다고 알려져 있다. 인간면역결핍 바이러스 (human immunodeficiency virus type-1, HIV-1)에 포함된 단백질인 Tat (trans-activator of transcription)은 세포 투과 기능이 관찰되며, 이러한 기능은 11개 아미노산으로 구성된 Tat 단백질의 중간부위인 단백질 형질도입 부위(protein transduction domain, PTD)의 특성 때문에 나타나며 아직 명확한 메카니즘은 알려지지 않았다. PTD이 규명된 이래 자연계에 존재하는 단백질로부터 유래하거나 인공적으로 디자인되어 합성한 다양한 CPP들이 보고되어 왔다(Frankel, A. D. et al., Cell. (1988) 55: 1189-1193). HIV-1 Tat은 양이온성의 짧은 도메인을 가지며 이후 연구들을 통해 이 도메인의 펩티드 서열(pTAT, YGRKKRRQRRR)이 규명되었다(Heitz, F. et al., Br. J. Pharmacol. (2009) 157: 195-206; Vives, E., P. et al., J. Biol. Chem. (1997) 272:16010-16017). 상기 TAT을 이용하여 FoxM1 점 돌연변이를 포함하는 펩티드를 세포내로 전달하여 전이성 암에 대한 억제 효과를 평가하고자 하였다.It is known that macromolecules such as proteins and nucleic acids are difficult to be delivered into cells because they cannot penetrate the cell membrane. Various technologies have been developed to deliver proteins into cells, and the most representative technology is cell-penetrating peptide (CPP). CPPs are short-length peptides consisting of between 8 and 30 amino acids and are classified as cationic, amphipathic, or hydrophobic. It is known to be able to pass through cell membranes. Tat (trans-activator of transcription), a protein contained in human immunodeficiency virus type-1 (HIV-1), is observed to have a cell-penetrating function, and this function is achieved through the middle region of the Tat protein, which consists of 11 amino acids. It appears due to the characteristics of the protein transduction domain (PTD), and the clear mechanism is not yet known. Since PTD was identified, various CPPs derived from proteins existing in nature or artificially designed and synthesized have been reported (Frankel, A. D. et al., Cell. (1988) 55: 1189-1193). HIV-1 Tat has a short cationic domain, and through subsequent studies, the peptide sequence (pTAT, YGRKKRRQRRR) of this domain was identified (Heitz, F. et al., Br. J. Pharmacol. (2009) 157: 195- 206; Vives, E., P. et al., J. Biol (1997) 272:16010-16017. Using the TAT, we attempted to evaluate the inhibitory effect on metastatic cancer by delivering a peptide containing a FoxM1 point mutation into cells.
먼저, FoxM1 인산화 점 돌연변이체 펩티드(QNAPAETSEE)를 세포내에 침투시키기 위한 CPP(Cell-Penetrating Peptide)의 한 종류인 TAT(YGRKKRRQRRR)를 붙여서 3개의 펩티드를 합성하였다(도 8A). 제작된 3개의 펩티드의 효능을 평가하기 위해서 caspase-3의 효소 활성을 이용한 세포사멸성과 migrationa assay를 이용한 세포이동성의 억제 효과를 관찰하였다(도 8B, 8C). A549 세포에 각각의 FoxM1 비인산화 점 돌연변이체 펩티드(5μM FoxM1-S25A peptide)를 48시간 처리후에 caspase-3의 효소 활성을 측정한 결과, 각각의 3개의 펩티드 처리에 의해서 세포사멸성이 증가함을 관찰할 수 있으며, 특히 펩티드#1과 펩티드#3에서 세포사멸성 효과가 크게 나타는 것을 확인할 수 있었다. 또한 A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 3개의 각각의 FoxM1 비인산화 점 돌연변이체 펩티드(5μM FoxM1-S25A peptide) 처리에 의해 암세포의 이동성의 억제 효과를 관찰하였다(도 8C). 3개의 펩티드 모두 암세포의 이동성 억제 효과를 관찰할 수 있었으며, 특히 펩티드#1에서 그 억제효과가 가장 큰 것을 관찰하였다.First, three peptides were synthesized by attaching TAT (YGRKKRRQRRR), a type of CPP (Cell-Penetrating Peptide), to infiltrate the FoxM1 phosphorylated point mutant peptide (QNAPAETSEE) into cells (Figure 8A). To evaluate the efficacy of the three produced peptides, the inhibitory effect on apoptosis using the enzymatic activity of caspase-3 and cell migration using a migration assay was observed (Figures 8B, 8C). As a result of measuring the enzyme activity of caspase-3 after treating A549 cells with each FoxM1 non-phosphorylated point mutant peptide (5 μM FoxM1-S25A peptide) for 48 hours, it was found that treatment with each of the three peptides increased apoptosis. It can be observed, and it was confirmed that the apoptotic effect was particularly significant in peptide #1 and peptide #3. Additionally, under conditions of overexpression of phosphorylated point mutants of FoxM1 in A549 cells, the inhibitory effect on cancer cell mobility was observed by treatment with each of the three non-phosphorylated FoxM1 point mutant peptides (5 μM FoxM1-S25A peptide) (Figure 8C). All three peptides were observed to have an inhibitory effect on the mobility of cancer cells, and in particular, peptide #1 had the greatest inhibitory effect.
본 발명자는 3개의 펩티드 중 가장 효능이 높다고 판단되는 펩티드#1의 세포내 침투여부를 알아보기 위해서 펩티드의 C 말단쪽에 FITC 형광물질을 붙여서 제작하였으며 (도 10A), 펩티드 처리 24시간 후에 펩티드가 세포내에 잘 투과됨을 관찰하였다(도 10B). TGF-β에 의해 유도된 암전이 조건에서 세포내로 투과된 펩티드의 처리가 상피간엽이행 마커(EMT marker)의 mRNA 발현 변화를 억제하는 것을 관찰하였다(도 10C). 추가적으로 FoxM1 비인산화 점 돌연변이 펩티드 처리가 TGF-β가 처리된 전이환경에서 증가된 암세포의 이동성를 억제하는 효과를 세포이동성 실험 (migration assay)을 통해서 확인할 수 있었다(도 10D). In order to determine whether peptide #1, which is considered to be the most effective among the three peptides, penetrates into cells, the present inventor attached FITC fluorescent material to the C-terminal side of the peptide to determine whether the peptide could penetrate the cell (Figure 10A). After 24 hours of peptide treatment, the peptide entered the cells. It was observed that it penetrated well (Figure 10B). It was observed that treatment with peptide permeated into cells under TGF-β-induced cancer metastasis conditions suppressed changes in mRNA expression of epithelial-mesenchymal transition marker (EMT marker) (Figure 10C). Additionally, the effect of treatment with FoxM1 non-phosphorylated point mutant peptide in suppressing the increased mobility of cancer cells in a metastatic environment treated with TGF-β was confirmed through a cell migration assay (Figure 10D).
또한 FoxM1 인산화 점 돌연변이체 단백질을 과발현시키는 폐암 세포 A549에 FoxM1 비인산화 점 돌연변이 펩티드 처리에 의해서 FoxM1의 과발현에 의해서 증가된 간엽이행 마커인 CDH2와 vimentin mRNA 발현이 감소하였으며, 반대로 상피형질 마커인 CDH1의 mRNA 발현 감소가 펩티드 처리에 의해서 증가되는 것을 관찰하였다(도 10E). 또한 A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 FoxM1 비인산화 점 돌연변이체 펩티드(5μM FoxM1-S25A peptide)처리에 의해 암세포의 이동성의 억제 효과를 관찰하였다(도 10F). In addition, treatment of lung cancer cells A549, which overexpress FoxM1 phosphorylated point mutant protein, with FoxM1 non-phosphorylated point mutant peptide decreased the expression of CDH2 and vimentin mRNA, which are mesenchymal transition markers, which were increased by overexpression of FoxM1. Conversely, the expression of CDH1, an epithelial marker, was decreased. We observed that the decrease in mRNA expression was increased by peptide treatment (Figure 10E). Additionally, under conditions of overexpression of a phosphorylated point mutant of FoxM1 in A549 cells, an inhibitory effect on the mobility of cancer cells was observed by treatment with non-phosphorylated FoxM1 point mutant peptide (5 μM FoxM1-S25A peptide) (Figure 10F).
추가적으로, FoxM1 비인산화 점 돌연변이체 펩티드가 종양관련 대식세포 분화성과 종양세포 면역회피능에 미치는 역할을 관찰하기 위해서 A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 FoxM1 비인산화 점 돌연변이체 펩티드(5μM FoxM1-S25A peptide)처리 후에 STAT1, VEGFA, c-fos, IL6, CD274 mRNA 변화를 관찰하였다. 펩티드 처리에 의해서 FoxM1의 과발현에 의해서 증가된 STAT1, VEGFA, c-fos, IL6, CD274 mRNA 발현이 억제되는 것을 관찰하였다(도 10G). 따라서 FoxM1 비인산화 점 돌연변이체 펩티드가 종양관련 대식세포 분화성과 종양세포 면역회피능에 억제효과가 있음을 확인할 수 있었다.Additionally, to observe the role of FoxM1 non-phosphorylated point mutant peptides on tumor-related macrophage differentiation and tumor cell immune evasion, FoxM1 non-phosphorylated point mutant peptides (5 μM FoxM1) were administered in A549 cells under overexpression conditions. After treatment (-S25A peptide), changes in STAT1, VEGFA, c-fos, IL6, and CD274 mRNA were observed. We observed that STAT1, VEGFA, c-fos, IL6, and CD274 mRNA expression, which was increased by overexpression of FoxM1, was suppressed by peptide treatment (Figure 10G). Therefore, it was confirmed that FoxM1 non-phosphorylated point mutant peptide had an inhibitory effect on tumor-related macrophage differentiation and tumor cell immune evasion ability.
아울러, 본 발명은 FoxM1 억제제 또는 비인산화 점 돌연변이를 포함하는 FoxM1 단백질 및 펩티드를 유효성분으로 포함하는 일차성 암 뿐만 아니라 전이성 암에 대한 항암 치료제를 제공한다. In addition, the present invention provides an anticancer treatment agent for primary cancer as well as metastatic cancer, comprising a FoxM1 inhibitor or a FoxM1 protein and peptide containing a non-phosphorylation point mutation as an active ingredient.
본 발명의 FoxM1 점 돌연변이를 포함하는 단백질 및 펩티드를 유효성분으로 포함하는 항암제는 FoxM1 의 발현이 과다한 다양한 암종 특히 폐암 (LIANG, Sheng-Kai, et al. Oncogene, 2021, 40.30: 4847-4858), 유방암 (ZIEGLER, Yvonne, et al. NPJ breast cancer, 2019, 5.1: 1-11), 간암 (YU, Chun-Peng, et al. Molecular medicine reports, 2017, 16.4: 5181-5188), 전립선암 (Kalin, Tanya V., et al. Cancer research 66.3 (2006): 1712-1720), 대장암 (Yoshida, Yuichi, et al. Gastroenterology 132.4 (2007): 1420-1431), 뇌암 (Liu, Mingguang, et al. Cancer research 66.7 (2006): 3593-3602) 등의 각종 고형암 및 백혈병 등의 예방 및 치료에 이용될 수 있으며(XU, Xin-Sen, et al. Asian Pacific Journal of Cancer Prevention, 2015, 16.1: 23-29.), 또한 일차암에서 전이되어 유발된 전이성 고형암의 예방 및 치료(WANG, Yi-Wei, et al. Journal of biomedical science, 2022, 29.1: 1-23)에도 이용될 수 있다.The anticancer agent containing the protein and peptide containing a FoxM1 point mutation of the present invention as an active ingredient is used to treat various carcinomas with excessive expression of FoxM1, especially lung cancer (LIANG, Sheng-Kai, et al. Oncogene, 2021, 40.30: 4847-4858), Breast cancer (ZIEGLER, Yvonne, et al. NPJ breast cancer, 2019, 5.1: 1-11), liver cancer (YU, Chun-Peng, et al. Molecular medicine reports, 2017, 16.4: 5181-5188), prostate cancer (Kalin , Tanya V., et al. Cancer research 66.3 (2006): 1712-1720), colon cancer (Yoshida, Yuichi, et al. Gastroenterology 132.4 (2007): 1420-1431), brain cancer (Liu, Mingguang, et al. Cancer research 66.7 (2006): 3593-3602) and can be used for the prevention and treatment of various solid cancers and leukemia (XU, Xin-Sen, et al. Asian Pacific Journal of Cancer Prevention, 2015, 16.1: 23- 29.), It can also be used for the prevention and treatment of metastatic solid cancer caused by metastasis from primary cancer (WANG, Yi-Wei, et al. Journal of biomedical science, 2022, 29.1: 1-23).
본원에 기재된 단백질 또는 폴리펩티드는 서열번호 1 또는 서열번호 2로 표시되는 아미노산 서열을 포함하거나, 서열번호 1 또는 서열번호 2와 실질적으로 동일한 생물학적 특성을 갖는, 80% 이상의 상동성, 85% 이상의 상동성, 86% 이상의 상동성, 87% 이상의 상동성, 88% 이상의 상동성, 89% 이상의 상동성, 90% 이상의 상동성, 91% 이상의 상동성, 92% 이상의 상동성, 93% 이상의 상동성, 94% 이상의 상동성, 95% 이상의 상동성, 96% 이상의 상동성, 97% 이상의 상동성, 98% 이상의 상동성, 또는 99% 이상의 상동성을 갖는 아미노산 서열을 포함하는 단백질 도는 폴리펩티드를 지칭한다. The protein or polypeptide described herein includes the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or has substantially the same biological properties as SEQ ID NO: 1 or SEQ ID NO: 2, at least 80% homology, at least 85% homology. , more than 86% homology, more than 87% homology, more than 88% homology, more than 89% homology, more than 90% homology, more than 91% homology, more than 92% homology, more than 93% homology, 94 Refers to a protein or polypeptide comprising an amino acid sequence with at least 95% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
본 기술분야에 알려진 바와 같이, 용어 "백분율 동일성(percent identity)"은 서열을 비교함으로써 결정된 바와 같이 둘 이상의 폴리펩티드 서열 또는 둘 이상의 폴리뉴클레오티드 서열 간의 관계이다. 또한, 본 기술분야에서, "동일성"은 경우에 따라, 상기 서열의 문자열 간의 일치에 의해 결정되는 것과 같이, 폴리펩티드 또는 폴리뉴클레오티드 서열간의 서열 관련 정도를 의미한다. "동일성" 및 "유사성"은 Computational Molecular Biology((Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991)에 기술된 것을 포함하나 이에 제한되지 않는, 알려진 방법들에 의해 쉽게 계산될 수 있다. 동일성을 결정하는 바람직한 방법은 시험한 서열 간의 최적의 일치를 제공하도록 고안된다. 동일성 및 유사성을 결정하는 방법은 공공이 이용가능한 컴퓨터 프로그램에서 성문화된다. 서열 정렬 및 백분율 동일성 계산은 레이져진 바이오인포매틱스 컴퓨팅 스위트(LASERGENE bioinformatics computing suite, DNASTAR Inc., Madison, WI)의 메갈린(Megalign) 프로그램과 같은 서열 분석 소프트웨어를 이용하여 수행될 수 있다. 서열의 다중 정렬(multiple alignment)은 기본 매개변수(default parameter)(GAP PENALTY=10, GAP LENGTH PENALTY=10)을 갖는 클러스탈(Clustal) 정렬 방법 (Higgins et al., CABIOS. 5:151 (1989))을 이용하여 수행될 수 있다. 클러스탈 방법을 이용한 쌍정렬(pairwise alignment)을 위한 기본 매개변수는 KTUPLE 1, GAP PENALTY=3, WINDOW=5 및 DIAGONALS SAVED=5로부터 선택될 수 있다. 당분야에 공지되어 있는 바와 같이, 2종의 폴리펩티드들 사이의 "유사성"은 아미노산 서열, 및 폴리펩티드의 보존된 아미노산 치환을 제2 폴리펩티드의 서열과 비교함으로써 측정한다. 본원에서 이러한 서열들에 대한 동일성 또는 상동성(homology)은 서열들을 정렬하고 필요하다면 갭(gap)을 도입하여 최대 상동성(%)을 달성하고 임의의 보존적 치환을 서열 동일성의 일부로서 간주하지 않은 후, 공지되어 있는 펩티드와 동일한, 후보 서열 내의 아미노산 잔기들의 백분율로서 정의된다. 펩티드 서열에서의 N-말단, C-말단 또는 내부 연장, 결실 또는 삽입은 상동성에 영향을 미치는 것으로서 해석되지 않을 것이다.As known in the art, the term “percent identity” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. Also, in the art, “identity” refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by correspondence between strings of said sequences. “Identity” and “similarity” are used in Computational Molecular Biology ((Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York ( 1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods for determining identity include sequence alignment and percent identity calculations, which are designed to provide optimal match between tested sequences. Multiple alignment of sequences can be performed using sequence analysis software, such as the Megalign program from the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), with default parameters. It can be performed using the Clustal alignment method (Higgins et al., CABIOS. 5:151 (1989)) with (default parameters) (GAP PENALTY=10, GAP LENGTH PENALTY=10). The default parameters for pairwise alignment using the method can be selected from KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. As is known in the art, “similarity” between two polypeptides is determined by comparing the amino acid sequences and conserved amino acid substitutions of the polypeptide to the sequence of the second polypeptide. Identity or homology to these sequences herein is defined by aligning the sequences and introducing gaps, if necessary, to achieve maximum percent homology and not considering any conservative substitutions as part of the sequence identity. It is defined as the percentage of amino acid residues in the candidate sequence that are identical to the known peptide. N-terminal, C-terminal or internal extensions, deletions or insertions in the peptide sequence will not be interpreted as affecting homology.
용어 "상동성(homology)"은 두 개의 폴리뉴클레오티드 또는 두 개의 폴리펩티드 부분들간의 동일성(identity)의 백분율을 지칭한다. 한 부분 내지 다른 부분의 서열 간의 관련성(correspondence)은 본 기술분야에 알려진 기술들에 의해 결정될 수 있다. 예를 들어, 상동성은 서열 정보를 정렬하여 그리고 즉시 이용가능한 컴퓨터 프로그램을 이용하여 두 개의 폴리펩티드 분자 간의 서열 정보를 직접적으로 비교함으로써 결정될 수 있다. 그렇지 않으면, 상동성은 동종 영역들 간에 안정적인 이중체(duplex)를 형성하는 조건 하에서 폴리뉴클레오티드들을 혼성화한 후, 단일 가닥 특이적 뉴클레아제를 이용하여 절단하고 절단된 단편들의 크기 결정에 의해 결정될 수 있다.The term “homology” refers to the percentage of identity between portions of two polynucleotides or two polypeptides. Correspondence between sequences from one part to another can be determined by techniques known in the art. For example, homology can be determined by aligning sequence information and directly comparing sequence information between two polypeptide molecules using readily available computer programs. Alternatively, homology can be determined by hybridizing the polynucleotides under conditions that form a stable duplex between homologous regions, followed by cleavage using a single-strand specific nuclease and size determination of the cleaved fragments. .
따라서, 모든 문법적 형태의 용어 "서열 유사성"은 공통의 진화적 기원을 갖거나 갖지 않을 수 있는 핵산 또는 아미노산 서열들 간의 동일성 또는 관련성 정도를 지칭한다(Reeck et al., Cell 50:667 (1987) 참조). 일 구체예에 있어서, 뉴클레오티드들의 약 50%(예컨대, 적어도 약 75%, 90%, 또는 95%)가 정의된 길이의 DNA 서열 이상 일치할 때, 두 개의 DNA 서열들은 "실질적으로 상동" 또는 "실질적으로 유사"하다. 실질적으로 상동인 서열들은 서열 데이터 뱅크 또는, 예를 들어, 특정 시스템을 위해 정의된 바와 같이 엄격한 조건하에서의 서던 혼성화 실험에서 이용가능한 표준 소프트웨어를 이용하여 서열을 비교함으로써 확인될 수 있다. 적절한 혼성화 조건을 정의하는 것은 본 기술분야의 기술 범주 내이다(예컨대, Sambrook et al., 1989, 참조).Accordingly, the term “sequence similarity” in all its grammatical forms refers to the degree of identity or relatedness between nucleic acid or amino acid sequences that may or may not have a common evolutionary origin (Reeck et al., Cell 50:667 (1987) reference). In one embodiment, two DNA sequences are “substantially homologous” or “when about 50% (e.g., at least about 75%, 90%, or 95%) of the nucleotides match at least over a defined length of DNA sequence. “substantially similar.” Substantially homologous sequences can be identified by comparing sequences using standard software available in sequence data banks or, for example, Southern hybridization experiments under stringent conditions as defined for the particular system. Defining appropriate hybridization conditions is within the skill of the art (see, e.g., Sambrook et al., 1989).
본 명세서에 사용된 바와 같이, "실질적으로 유사"는 하나 이상의 뉴클레오티드 염기의 변화가 하나 이상의 아미노산의 치환을 야기하지만 상기 DNA 서열에 의해 암호화되는 단백질의 기능적 특성에는 영향을 미치는 않는 핵산 단편을 지칭한다. "실질적으로 유사"는 또한 하나 이상의 뉴클레오티드 염기의 변화가 안티센스 또는 동시억제(co-suppression) 기법에 의해 유전자 발현의 변화를 매개하는 핵산 단편의 능력에 영향을 미치지 않는 핵산 단편을 지칭한다. "실질적으로 유사"는 또한 얻어지는 전사체의 기능적 특성에는 실질적으로 영향을 주지 않는 하나 이상의 뉴클레오티드 염기의 결손 또는 삽입과 같은, 본 발명의 핵산 단편의 변형을 지칭한다. 따라서, 본 발명은 예시된 특정 서열보다 더 포함한다고 이해된다. 제안된 각각의 변형들은 암호화된 생성물의 생물학적 활성의 유지(retention)를 결정하는 것과 같이, 본 기술분야에서 통상적인 기술 내에 속한다.As used herein, “substantially similar” refers to a nucleic acid fragment in which a change in one or more nucleotide bases results in the substitution of one or more amino acids but does not affect the functional properties of the protein encoded by said DNA sequence. . “Substantially similar” also refers to a nucleic acid fragment in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate changes in gene expression by antisense or co-suppression techniques. “Substantially similar” also refers to modifications of a nucleic acid fragment of the invention, such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. Accordingly, it is understood that the invention encompasses more than the specific sequences exemplified. Each of the proposed modifications is within the ordinary skill in the art as it determines retention of biological activity of the encoded product.
나아가, 숙련된 당업자는 엄격한 조건(0.1X SSC, 0.1% SDS, 65℃ 및 2X SSC, 0.1% SDS로 세척 후, 0.1X SSC, 0.1% SDS) 하에서 본 명세서에 예시된 서열들과 혼성화하는 능력에 의해 본 발명에 의해 포함된 실질적으로 유사한 서열들이 정의된다는 것을 인식한다. 실질적으로 유사한 본 발명의 핵산 단편들은 DNA 서열들이 본 명세서에 보고된 핵산 단편의 DNA 서열과 적어도 약 70%, 80%, 90% 또는 95% 동일한 핵산 단편들이다.Furthermore, one skilled in the art will recognize the ability to hybridize with the sequences exemplified herein under stringent conditions (0.1 It is recognized that substantially similar sequences encompassed by the present invention are defined by . Substantially similar nucleic acid fragments of the invention are nucleic acid fragments whose DNA sequences are at least about 70%, 80%, 90% or 95% identical to the DNA sequence of the nucleic acid fragment reported herein.
폴리펩티드 또는 단백질의 "변형체(variant)"는 폴리펩티드 또는 단백질로부터 유래하고 상기 폴리펩티드 또는 단백질의 적어도 하나의 생물학적 특성을 유지하고 있는 임의의 유사체, 단편, 유도체 또는 돌연변이를 지칭한다. 상기 폴리펩티드 또는 단백질의 상이한 변형체들이 자연 내에 존재할 수 있다. 이들 변형체들은 단백질을 암호화하는 구조 유전자의 뉴클레오티드 서열이 다른 것에 특징이 있는 대립 유전자의 변이일 수 있거나, 차별적 스플라이싱 또는 번역 후 수식(modification)을 포함할 수 있다. 숙련된 당업자는 하나 또는 복수의 아미노산 치환, 결손, 부가 또는 대체를 갖는 변형체를 생성할 수 있다. 이들 변형체들은 그중에서도: (a) 하나 이상의 아미노산 잔기들이 보존적 또는 비보존적 아미노산들로 치환된 변형체, (b) 하나 이상의 아미노산이 폴리펩티드 또는 단백질에 부가된 변형체, (c) 아미노산 중 하나 이상이 치환기를 포함하는 변형체, 및 (d) 폴리펩티드 또는 단백질이 혈청 알부민과 같은 또 다른 폴리펩티드와 융합된 변형체를 포함할 수 있다. A “variant” of a polypeptide or protein refers to any analog, fragment, derivative or mutation derived from the polypeptide or protein and retaining at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequence of the structural gene encoding the protein, or may involve differential splicing or post-translational modifications. A skilled artisan can create variants with one or more amino acid substitutions, deletions, additions or substitutions. These variants include, inter alia: (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to a polypeptide or protein, (c) variants in which one or more of the amino acids is a substituent. and (d) variants in which a polypeptide or protein is fused with another polypeptide, such as serum albumin.
또한 보존적 변형체는 단백질의 생물학적 기능에 불리한 영향을 미치지 않는 서열 변경을 가진 아미노산 서열을 의미한다. 변경된 서열이 단백질과 관련된 생물학적 기능을 방해하거나 파괴하는 경우, 치환, 삽입 또는 결실이 단백질에 불리한 영향을 미친다고 기재된다. 예를 들어, 단백질의 총 전하, 구조 또는 소수성-친수성은 생물학적 활성에 불리한 영향을 미치지 않으면서 변경될 수 있다. 따라서, 아미노산 서열은 예를 들어, 단백질의 생물학적 활성에 불리한 영향을 미치지 않으면서 펩티드가 보다 높은 소수성 또는 친수성을 나타내도록 변경될 수 있다. 유전적(억제, 결손, 돌연변이 등), 화학적 및 효소적 기술들을 포함하는, 이러한 변형체를 수득하는 기술들이 본 기술분야에서 통상의 기술을 가진 자에게 알려져 있다.Conservative variants also refer to amino acid sequences with sequence changes that do not adversely affect the biological function of the protein. A substitution, insertion, or deletion is said to have an adverse effect on a protein if the altered sequence interferes with or destroys the biological function associated with the protein. For example, the total charge, structure, or hydrophobicity-hydrophilicity of a protein can be altered without adversely affecting its biological activity. Thus, the amino acid sequence can be altered, for example, to make the peptide more hydrophobic or hydrophilic without adversely affecting the biological activity of the protein. Techniques for obtaining such variants are known to those skilled in the art, including genetic (suppression, deletion, mutation, etc.), chemical and enzymatic techniques.
"보존적(conservative) 아미노산 치환"은 아미노산 잔기가 유사한 곁사슬(side chain)을 갖는 아미노산 잔기로 대체되는 것이다. 유사한 곁사슬을 갖는 아미노산 잔기들의 패밀리는 본 기술분야 내에서 정의되어 왔다. 이들 패밀리는 염기성 결사슬을 갖는 아미노산(예컨대, Lys, Arg, His), 산성 곁사슬을 갖는 아미노산(예컨대, Asp, Glu), 전하를 띠지 않는 극성 곁사슬을 갖는 아미노산(예컨대, Gly, Asn, Gln, Ser, Thr, Tyr, Cys), 비극성 곁사슬을 갖는 아미노산(예컨대, Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), 베타-가지의 곁사슬을 갖는 아미노산(예컨대, Thr, Val, Ile) 및 방향족 곁사슬을 갖는 아미노산(예컨대, Tyr, Phe, His)을 포함한다.A “conservative amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined within the art. These families include amino acids with basic chains (e.g., Lys, Arg, His), amino acids with acidic side chains (e.g., Asp, Glu), and amino acids with uncharged polar side chains (e.g., Gly, Asn, Gln, Ser, Thr, Tyr, Cys), amino acids with nonpolar side chains (e.g., Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), amino acids with beta-branched side chains (e.g., Thr, Val, Ile ) and amino acids with aromatic side chains (e.g., Tyr, Phe, His).
단백질 또는 폴리펩티드는 인산화와 같은 번역 후 수식 과정을 거칠 수 있다. 단백질 인산화는 가역적 과정이며 단백질 키나아제에 의해 촉매된다. 포유동물에서 대부분의 인산화는 단백질 또는 아마노산 중 Ser, Thr, 또는 Tyr 잔기에서 발생된다. 본 발명의 일 실시예에서, 단백질 인산화을 억제하기 위하여, 인산화 잔기인 Ser을 비인산화 아미노산인 Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His, 또는 Lys으로 치환할 수 있고, 본 발명의 일 실시예에서는 Gly, Ala, val, 또는 Cys으로 치환될 수 있다. 본 발명의 일 실시예에서 Ser은 Ala으로 치환된다. Proteins or polypeptides may undergo post-translational modification processes such as phosphorylation. Protein phosphorylation is a reversible process and is catalyzed by protein kinases. In mammals, most phosphorylation occurs on Ser, Thr, or Tyr residues in proteins or amino acids. In one embodiment of the present invention, in order to inhibit protein phosphorylation, the phosphorylated residue Ser is replaced with non-phosphorylated amino acids Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His. , or Lys, and in one embodiment of the present invention, it may be substituted with Gly, Ala, val, or Cys. In one embodiment of the present invention, Ser is replaced with Ala.
DNA "암호화 서열"은 폴리펩티드를 암호화하고 적절한 조절 서열의 통제하에 놓여졌을 때 시험관 내(in vitro) 또는 생체 내(in vivo) 세포에서 폴리펩티드로 전사 및 번역될 수 있는 이중 가닥 DNA 서열을 지칭한다. "적절한 조절 서열"은 암호화 서열의 상류(5' 비암호화 서열), 내부, 또는 하류(3' 비암호화 서열)에 위치하는 뉴클레오티드 서열을 지칭하며, 이는 연관된 암호화 서열의 전사, RNA 가공 또는 안정성 또는 번역에 영향을 미친다. 조절 서열에는 프로모터, 번역 리더 서열, 인트론, 폴리아데닐레이션 인식 서열, RNA 가공 부위, 작동자(effector) 결합부위 및 줄기-루프(stem-loop) 구조가 포함될 수 있다. 상기 암호화 서열의 경계는 5'(아미노) 말단의 개시 코돈 및 3'(카르복실) 말단의 번역 종결 코돈에 의해 결정된다. 암호화 서열은 원핵 서열, mRNA로부터의 cDNA, 게놈 DNA 서열 및 심지어 합성 DNA 서열을 포함할 수 있으나, 이에 제한되지 않는다. 만약 상기 암호화 서열을 진핵 세포에서 발현시키고자 한다면, 폴리아데닐레이션 신호 및 전사 종결 서열이 일반적으로 상기 암호화 서열의 3'쪽에 위치할 것이다.DNA “coding sequence” refers to a double-stranded DNA sequence that encodes a polypeptide and can be transcribed and translated into a polypeptide in cells in vitro or in vivo when placed under the control of appropriate regulatory sequences. "Appropriate regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding sequence, which may be used to control transcription, RNA processing, or stability of the associated coding sequence. Affects translation. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of the coding sequence are determined by an initiation codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences may include, but are not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is to be expressed in eukaryotic cells, the polyadenylation signal and transcription termination sequence will generally be located on the 3' side of the coding sequence.
본 발명에서 용어, 본 발명에서, "재조합 벡터"란 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 발현 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 말한다.As used herein, the term "recombinant vector" refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express the gene insert.
용어 "플라스미드"는 흔히 세포의 주요 기작의 일부가 아닌 유전자를 운반하고, 보통 원형 이중-가닥 DNA 분자의 형태로 존재하는 염색체외 요소를 지칭한다. 그러한 요소들은 임의의 소스(source)로부터 유래된, 자율적 복제 서열, 유전자 통합 서열, 파지 또는 뉴클레오티드 서열, 선형, 원형 또는 고차나선형(supercoil)의 단일- 또는 이중-가닥 DNA 또는 RNA일 수 있으며, 여기에서 수많은 뉴클레오티드 서열들이 연결되거나 재조합되어 프로모터 단편 및 선택된 유전자 산물을 위한 DNA 서열을 적절한 3'미번역 서열과 함께 세포 내로 도입할 수 있는 독특한 구조체를 형성한다. 일반적으로, 플라스미드는 박테리아 숙주 세포(예를 들면, 대장균(Escherichia coli))에서 기능을 하는 복제 기원, 및 플라스미드를 포함하는 박테리아 숙주 세포의 검출용 선택가능한 마커를 포함한다.The term “plasmid” refers to an extrachromosomal element that often carries genes that are not part of the cell's main machinery and usually exists in the form of a circular double-stranded DNA molecule. Such elements may be autonomously replicating sequences, genetic integration sequences, phage or nucleotide sequences, linear, circular or supercoiled single- or double-stranded DNA or RNA, from any source, where: Numerous nucleotide sequences are linked or recombined to form a unique construct that can introduce the promoter fragment and DNA sequence for the selected gene product into the cell along with the appropriate 3'untranslated sequence. Typically, a plasmid includes an origin of replication that functions in a bacterial host cell (e.g., Escherichia coli), and a selectable marker for detection of the bacterial host cell containing the plasmid.
용어 "발현 벡터"는 삽입된 핵산 서열을 발현한 후 숙주를 형질전환시킬 수 있도록 고안된 벡터, 플라스미드 또는 운반체를 지칭한다. 클론된 유전자, 즉, 삽입된 핵산 서열은 일반적으로 프로모터, 최소 프로모터, 인핸서 등과 같은 조절 요소의 통제 하에 놓여진다. 원하는 숙주 세포에서의 핵산의 발현을 유도하는데 유용한 개시 조절 영역 또는 프로모터들은 무수히 많으며 본 기술분야의 숙련자들에게 잘 알려져 있다. 실질적으로 이들 유전자들의 발현을 유도할 수 있는 임의의 프로모터는 바이러스 프로모터, 박테리아 프로모터, 동물 프로모터, 포유동물 프로모터, 합성 프로모터, 구성 프로모터, 조직 특이적 프로모터, 발병(pathogenesis) 또는 질병 관련 프로모터, 발생 특이적 프로모터(developmental specific promoter), 유도성 프로모터(inducible promoter), 약하게 조절된 프로모터(light regulated promoter)를 포함하나, 이에 제한되지는 않으며; SV40 초기(SV40) 프로모터 영역, 라우스 육종 바이러스(Rous sarcoma virus, RSV)의 3' 긴 말단 반복(LTR)에 함유된 프로모터, 아데노바이러스(Ad)의 E1A 또는 주요 후기 프로모터(major late promoter, MLP), 인간 사이토메갈로바이러스 (human cytomegalovirus, HCMV) 즉시 초기 프로모터(immediate early promoter), 단순 포진 바이러스(HSV) 티미딘 키나아제(TK) 프로모터, 배큘로바이러스 IE1 프로모터, 신장 인자(elongation factor) 1 알파(EF1) 프로모터, 글리세르알데하이드-3-포스페이트 탈수소효소(GSPDH) 프로모터, 포스포글리세레이트 키나아제(PGK) 프로모터, 유비퀴틴 C(Ubc) 프로모터, 알부민 프로모터, 마우스 메탈로티오네인(metallothionein)-L 프로모터 및 전사 조절 영역의 조절 서열, 유비쿼터스 프로모터(HPRT, 비멘틴(vimentin), β-액틴, 튜불린 등), 중간필라멘트(intermediate filament)(데스민(desmin), 신경미세섬유, 케라틴, GFAP 등), 치료 유전자의 프로모터(MDR, CFTR 또는 인자 VIII 형태 등의), 발병 또는 질병 관련 프로모터, 및 췌장 선방세포(pancreatic acinar cell) 에서 활성인 엘라스타제(elastase) I 유전자 조절 영역과 같은, 조직 특이성을 나타내며 형질전환 동물에서 이용되어 온 프로모터; 췌장 베타 세포에서 활성인 인슐린 유전자 조절 영역, 림프계(lymphoid) 세포에서 활성인 면역글로불린 유전자 조절 영역, 고환, 유방, 림프계 및 대식 세포에서 활성인 마우스 유방암 바이러스 조절 영역; 간에서 활성인 알부민 유전자, Apo AI 및 Apo AII 조절 영역, 간에서 활성인 알파-태아단백질(fetoprotein) 유전자 조절 영역, 간에서 활성인 알파1-안티트립신 유전자 조절 영역, 골수 세포에서 활성인 베타-글로빈 유전자 조절 영역, 뇌에서의 희소돌기아교세포(oligodendrocyte cell)에서 활성인 마이엘린 염기성 단백질(myelin basic protein) 조절 영역, 골격근에서 활성인 미오신 경쇄-2 유전자 조절 영역 및 시상하부에서 활성인 생식선 자극호르몬 방출 호르몬 (gonadotropic releasing hormone), 피루브산 키나아제 프로모터, 빌린(villin) 프로모터, 지방산 결합 장내 단백질의 프로모터, 평활근 세포 β-액틴의 프로모터 등을 포함하나, 이에 제한되지 않는다.The term “expression vector” refers to a vector, plasmid, or carrier designed to express an inserted nucleic acid sequence and then transform a host. The cloned gene, i.e., the inserted nucleic acid sequence, is generally placed under the control of regulatory elements such as promoters, minimal promoters, enhancers, etc. There are numerous initiation control regions or promoters useful for directing expression of a nucleic acid in a desired host cell and are well known to those skilled in the art. Substantially any promoter capable of driving the expression of these genes may include viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue-specific promoters, pathogenesis- or disease-related promoters, and development-specific promoters. Including, but not limited to, developmental specific promoters, inducible promoters, and light regulated promoters; SV40 early (SV40) promoter region, promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), E1A or major late promoter (MLP) of adenovirus (Ad) , human cytomegalovirus (HCMV) immediate early promoter, herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) ) promoter, glyceraldehyde-3-phosphate dehydrogenase (GSPDH) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin C (Ubc) promoter, albumin promoter, mouse metallothionein-L promoter and transcription. Regulatory sequences in the regulatory region, ubiquitous promoters (HPRT, vimentin, β-actin, tubulin, etc.), intermediate filaments (desmin, neurofilament, keratin, GFAP, etc.), treatment Shows tissue specificity, such as promoters of genes (such as MDR, CFTR, or factor VIII forms), pathogenesis- or disease-related promoters, and elastase I gene regulatory regions that are active in pancreatic acinar cells. Promoters that have been used in transgenic animals; an insulin gene regulatory region active in pancreatic beta cells, an immunoglobulin gene regulatory region active in lymphoid cells, a mouse breast cancer virus regulatory region active in testicular, breast, lymphoid and macrophage cells; Albumin gene, Apo AI and Apo AII regulatory regions active in the liver, alpha-fetoprotein gene regulatory region active in the liver, alpha1-antitrypsin gene regulatory region active in the liver, beta-active in bone marrow cells. Globin gene regulatory domain, myelin basic protein regulatory domain active in oligodendrocyte cells in the brain, myosin light chain-2 gene regulatory domain active in skeletal muscle, and gonadal stimulation active in the hypothalamus. It includes, but is not limited to, gonadotropic releasing hormone, pyruvate kinase promoter, villin promoter, fatty acid binding intestinal protein promoter, smooth muscle cell β-actin promoter, etc.
"핵산", "핵산 분자", "올리고뉴클레오티드" 및 "폴리뉴클레오티드"는 상호교환적으로 사용되고, 단일 가닥 형태나 이중 가닥 나선(helix)으로의 리보뉴클레오사이드(아데노신, 구아노신, 우리딘 또는 시티딘; "RNA 분자") 또는 데옥시리보뉴클레오사이드(데옥시아데노신, 데옥시구아노신, 데옥시티민 또는 데옥시시티딘; "DNA 분자")의 인산 에스테르의 중합체 형태 또는 포스포로티오에이트(phosphorothioate) 및 티오에스테르와 같은 이의 임의의 인산 에스테르 유사체를 지칭한다. 이중 나선 DNA-DNA, DNA-RNA 및 RNA-RNA 나선이 가능하다. 용어 핵산 분자, 및 특히 DNA 또는 RNA 분자는 상기 분자의 일차 및 이차 구조만을 지칭하며 어느 특정 삼차 형태에 제한하지는 않는다. 따라서, 이 용어는 그 중에서도 선형 또는 환형 DNA 분자(예컨대, 제한효소 단편), 플라스미드, 초나선형(supercoiled) DNA 및 염색체로 발견되는 이중 가닥 DNA를 포함한다. 특정 이중 가닥 DNA 분자의 구조를 논할 때, 서열을 전사되지 않은 DNA 가닥(즉, mRNA에 일치하는 서열을 갖는 가닥)을 따라 5'에서 3' 방향으로만 제시하는 일반적인 규약에 따라 본 명세서에 서열이 기술될 수 있다. "재조합 DNA 분자"는 분자 생물학적 조작을 거친 DNA 분자이다. DNA는 cDNA, 게놈 DNA, 플라스미드 DNA, 합성 DNA 및 반합성 DNA를 포함하나, 이에 제한되지 않는다.“Nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” and “polynucleotide” are used interchangeably and refer to ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecule”) or a polymeric form of a phosphate ester of a deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxycytimine or deoxycytidine; “DNA molecule”) or phosphorothioate. (phosphorothioate) and any of its phosphoric acid ester analogues, such as thioesters. Double helix DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and especially DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Accordingly, the term includes double-stranded DNA found in linear or circular DNA molecules (e.g., restriction enzyme fragments), plasmids, supercoiled DNA, and chromosomes, among others. When discussing the structure of a particular double-stranded DNA molecule, the sequence is presented herein in accordance with the general convention of presenting the sequence only in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand with the sequence matching the mRNA). This can be described. A “recombinant DNA molecule” is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.
용어 "형질감염(transfection)"은 세포에 의한 외인성 또는 이종(heterologous) RNA 또는 DNA의 흡수(uptake)를 지칭한다. 외인성 또는 이종 RNA 또는 DNA가 세포 내부로 도입될 때, 이러한 RNA 또는 DNA에 의해 세포가 "형질감염"된다. 상기 형질감염된 RNA 또는 DNA가 표현형(phenotypic)의 변화를 가져올 때, 외인성 또는 이종 RNA 또는 DNA에 의해 세포는 "형질전환"된다. 상기 형질전환시키는 RNA 또는 DNA는 염색 DNA 내로 삽입(공유 결합)되어 세포의 게놈을 구성할 수 있다.The term “transfection” refers to the uptake of exogenous or heterologous RNA or DNA by a cell. When exogenous or foreign RNA or DNA is introduced into a cell, the cell is “transfected” by such RNA or DNA. A cell is “transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA results in a phenotypic change. The transforming RNA or DNA can be inserted (covalently linked) into the dyed DNA to construct the genome of the cell.
용어 "발현"은 코딩 서열에 의해 코딩된 생성물의 생물학적 생산을 지칭한다. 대부분의 경우에서, 코딩 서열을 포함하는 DNA 서열은 전사되어 메신저-RNA(mRNA)를 형성한다. 이어서, 상기 메신저 RNA는 번역되어 관련 생물학적 활성을 갖는 폴리펩티드 생성물을 형성한다. 또한, 발현 과정은 RNA 전사 생성물에 대한 추가 가공 단계(예를 들면, 인트론을 제거하기 위한 스플라이싱), 및/또는 폴리펩티드 생성물의 번역-후 가공을 포함할 수 있다.The term “expression” refers to the biological production of a product encoded by a coding sequence. In most cases, the DNA sequence containing the coding sequence is transcribed to form messenger-RNA (mRNA). The messenger RNA is then translated to form a polypeptide product with relevant biological activity. Additionally, the expression process may include additional processing steps for the RNA transcription product (e.g., splicing to remove introns), and/or post-translational processing of the polypeptide product.
본 발명은 본 발명의 벡터를 포함하는 숙주 세포를 제공한다. 숙주 세포는 원핵(예를 들어, 박테리아) 및 진핵 (예를 들어, 곰팡이, 효모, 동물, 곤충, 식물) 세포를 포함하며 융합 단백질의 발현에 적절한 임의의 세포일 수 있다. 적절한 원핵 숙주 세포는 E.coli(예를 들어, DH5, HB101, JM109 또는 W3110 계통), 바실루스(Bacillus), 스트렙토마이시스(Streptomyces), 살모넬라(Salmonella), 세라티아(Serratia) 및 슈도모나스(Pseudomonas) 종을 포함하나 이에 한정되지 않는다. 적절한 진핵 숙주 세포는 COS, CHO, HepG-2, CV-1, LLCMK2, 3T3, HeLa, RPMI8226, 293, BHK-21, Sf9, 사카로마이세스, 피치아(Pichia), 한세눌라(Hansenula), 클루이베로마이세스(Kluyveromyces), 아스페르길루스(Aspergillus) 또는 트리코데르마(Trichoderma) 종을 포함하나 이에 한정되지 않는다.The present invention provides host cells containing the vector of the present invention. Host cells include prokaryotic (e.g., bacteria) and eukaryotic (e.g., fungi, yeast, animals, insects, plants) cells and can be any cell suitable for expression of the fusion protein. Suitable prokaryotic host cells include E. coli (e.g., strains DH5, HB101, JM109, or W3110), Bacillus, Streptomyces, Salmonella, Serratia, and Pseudomonas. Including, but not limited to, species. Suitable eukaryotic host cells include COS, CHO, HepG-2, CV-1, LLCMK2, 3T3, HeLa, RPMI8226, 293, BHK-21, Sf9, Saccharomyces, Pichia, Hansenula, Including, but not limited to, Kluyveromyces, Aspergillus, or Trichoderma species.
본원에 기술된 펩티드, 단백질, 폴리펩티드, 핵산, siRNA, shRNA, 또는 miRNA 및 추가적인 물질, 예를 들어, 면역 체크포인트 저해제는 치료학적 또는 예방학적 치료를 위한 약학 조성물 또는 약제로 투여될 수 있으며, 약학적으로 허용되는 담체를 포함할 수 있고, 선택적으로 하나 이상의 보강제, 안정화제 등을 포함할 수 있는 임의의 적합한 약학적 조성물의 형태로 투여될 수 있다. 일 구현예에서, 약학 조성물은 치료학적 또는 예방학적 치료, 예를 들어 본원에 기술된 것들과 같은 암 질환과 같은 질환을 치료 또는 예방하는 데에 사용하기 위한 것이다. The peptides, proteins, polypeptides, nucleic acids, siRNAs, shRNAs, or miRNAs described herein and additional agents, e.g., immune checkpoint inhibitors, can be administered as pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment. It may be administered in the form of any suitable pharmaceutical composition, which may include an acceptable carrier and optionally one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is for use in therapeutic or prophylactic treatment, e.g., treating or preventing a disease, such as a cancer disease, such as those described herein.
용어 "약학 조성물"은 치료학적으로 유효한 물질을, 바람직하게는 약제학적으로 허용가능한 담체, 희석제 및/또는 부형제와 함께, 포함하는 제형에 관한 것이다. 상기 약학 조성물은, 상기 약학 조성물을 개체에 투여함으로써 질환 또는 장애의 중증도를 낮추거나, 예방하거나 또는 치료하는 데에 유용하다. 약학 조성물은 또한 약학적 제형으로서 당해 기술 분야에 공지되어 있다. 본 발명의 맥락에서, 약학 조성물은 본원에 기술된 바와 같이 펩티드, 단백질, 폴리펩티드, RNA, 또는 RNA 입자를 포함한다.The term “pharmaceutical composition” relates to a formulation comprising therapeutically effective substances, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical composition is useful for reducing the severity of, preventing, or treating a disease or disorder by administering the pharmaceutical composition to an individual. Pharmaceutical compositions are also known in the art as pharmaceutical formulations. In the context of the present invention, pharmaceutical compositions include peptides, proteins, polypeptides, RNA, or RNA particles as described herein.
본 명세서의 약학 조성물은 하나 이상의 보강제를 포함할 수 있거나, 하나 이상의 보강제와 함께 투여될 수 있다. 용어 "보강제"는 면역 반응을 연장, 강화 또는 가속화하는 화합물을 지칭한다. 보강제는 오일 에멀젼 (예, 프로인드 보강제), 미네랄 화합물 (예, 알럼), 박테리아 생산물 (예, 백일해균 독소) 또는 면역-자극 복합체와 같은 이종적인 화합물 군을 포함한다. 보강제의 예로는, 비-제한적으로, LPS, GP96, CpG 올리고데옥시뉴클레오티드, 성장인자 및 모노카인, 림포카인, 인터루킨, 케모카인과 같은 사이토카인을 포함한다. 사이토카인은 IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a일 수 있다. 추가적으로 알려진 보강제는 알루미늄 하이드록사이드, 프로인드 보강제 또는 Montanide® ISA51과 같은 오일이다. 본 명세서에서 사용하기 위한 적합한 다른 보강제는 Pam3Cys과 같은 리포펩티드를 포함한다.The pharmaceutical composition herein may contain one or more adjuvants or may be administered together with one or more adjuvants. The term “adjuvant” refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (eg Freund's adjuvant), mineral compounds (eg alum), bacterial products (eg pertussis toxin) or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, and chemokines. The cytokine may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Additional known adjuvants are aluminum hydroxide, Freund's adjuvant or oils such as Montanide® ISA51. Other adjuvants suitable for use herein include lipopeptides such as Pam3Cys.
본 명세서에 따른 약학 조성물은 일반적으로 "약제학적 유효량"으로, "약제학적으로 허용가능한 제제"로 적용된다.The pharmaceutical composition according to the present specification is generally applied as a “pharmaceutically effective amount” and as a “pharmaceutically acceptable formulation.”
용어 "약제학적으로 허용가능한"은 약학 조성물의 활성 성분의 작용과 상호작용하지 않는 물질의 무독성을 의미한다.The term “pharmaceutically acceptable” refers to the non-toxic nature of a substance that does not interact with the action of the active ingredients of the pharmaceutical composition.
용어 "약제학적 유효량" 또는 "치료학적 유효량"은 단독으로 또는 추가적인 투여와 더불어 원하는 반응 또는 원하는 효과를 달성하는 양을 의미한다. 특정 질환을 치료하는 경우에, 원하는 반응은 바람직하게는 질환의 진행 저해를 의미하다. 이는 질환의 진척 속도를 늦추는 것을 포함하며, 특히 질환의 진척을 중단시키거나 또는 역전시키는 것을 포함한다. 질환의 치료에서 원하는 반응은 또한 상기 질환 또는 상기 병태의 개시 지연 또는 개시 예방일 수 있다. 본원에 기술된 조성물의 유효량은 치료할 병태, 질환의 중등도, 나이, 신체 상태, 키 및 체중을 포함하는 환자의 개별 특성, 치료 기간, (존재하는 경우) 동반되는 요법의 유형, 구체적인 투여 경로 및 유사 인자에 따라 결정될 것이다. 따라서, 본원에 기술된 조성믈의 투여 용량은 이러한 다양한 특성에 따라 결정될 수 있다. 환자에서 반응이 1차 투여로 충분하지 않을 경우, 이 보다 고 용량 (또는 효과적으로는 다른, 보다 국지적인 투여 경로에 의해 달성되는 더 높은 용량)이 사용될 수 있다.The term “pharmaceutically effective amount” or “therapeutically effective amount” means the amount that, alone or in combination with additional administration, achieves the desired response or desired effect. When treating a particular disease, the desired response preferably means inhibition of progression of the disease. This includes slowing down the progression of the disease, and in particular stopping or reversing the progression of the disease. The desired response in the treatment of a disease may also be delaying the onset or preventing the onset of the disease or condition. The effective amount of the composition described herein will depend on the condition being treated, the severity of the disease, the individual characteristics of the patient, including age, physical condition, height and weight, the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and the like. It will be decided depending on factors. Accordingly, the dosage of the compositions described herein can be determined according to these various properties. If the response in the patient is not sufficient with the first dose, higher doses (or effectively higher doses achieved by other, more localized routes of administration) may be used.
본 명세서의 약학적 조성물은 염, 완충제, 보존제, 그리고 선택적으로 다른 치료학적 물질을 포함할 수 있다. 일 구현예에서, 본 명세서의 약학적 조성물은 하나 이상의 약학적으로 허용되는 담체, 희석제 및/또는 부형제를 포함한다.Pharmaceutical compositions herein may include salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions herein include one or more pharmaceutically acceptable carriers, diluents and/or excipients.
본 명세서의 약학 조성물에 사용하기 적합한 보존제는, 비-제한적으로, 벤즈알코늄 클로라이드, 클로로부탄올, 파라벤 및 티메로살을 포함한다.Preservatives suitable for use in the pharmaceutical compositions herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
본원에서 사용되는 바와 같이, 용어 "부형제"는 본 명세서의 약학 조성물에 존재할 수 있지만 활성 성분이 아닌 물질을 지칭한다. 부형제에 대한 예로는, 비-제한적으로, 담체, 결합제, 희석제, 윤활제, 증점제, 계면활성제, 보존제, 안정화제, 유화제, 완충제, 착향제 또는 착색제를 포함한다.As used herein, the term “excipient” refers to a substance that may be present in the pharmaceutical compositions herein but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.
용어 "희석제"는 희석 및/또는 묽게 하는 물질을 의미한다. 또한, 용어 "희석제"는 유체, 액체 또는 고체 현탁물 및/또는 혼합 매질 중 어느 하나 이상을 포함한다. 적합한 희석제의 예는 에탄올, 글리세롤 및 물을 포함한다.The term “diluent” means a substance that dilutes and/or thins. Additionally, the term “diluent” includes any one or more of fluids, liquids or solid suspensions and/or mixed media. Examples of suitable diluents include ethanol, glycerol, and water.
용어 "담체"는 약학 조성물의 투여를 쉽게 만들거나, 강화하거나 또는 투여를 수행할 수 있게 하기 위해 활성 성분과 조합되는 천연, 합성, 유기, 무기일 수 있는 성분을 지칭한다. 본원에서 사용된 바와 같이, 담체는, 개체에 투여하기 적합한, 하나 이상의 혼용가능한 고체 또는 액체 충전제, 희석제 또는 캡슐화 물질일 수 있다. 적합한 담체로는, 비-제한적으로, 멸균수, 링거, 링거 락테이트, 멸균 소듐 클로라이드 용액, 등장성 식염수, 폴리알킬렌 글리콜, 수소화 나프탈렌, 및, 특히, 생체적합성 락티드 폴리머, 락티드/글리콜라이드 코폴리머 또는 폴리옥시에틸렌/폴리옥시-프로필렌 코폴리머를 포함한다. 일 구현예에서, 본 명세서의 약학 조성물은 등장성 식염수를 포함한다.The term “carrier” refers to a substance, which may be natural, synthetic, organic, or inorganic, that is combined with the active ingredient to facilitate, enhance, or facilitate administration of the pharmaceutical composition. As used herein, a carrier can be one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, especially, biocompatible lactide polymers, lactide/glycols. ride copolymers or polyoxyethylene/polyoxy-propylene copolymers. In one embodiment, the pharmaceutical composition herein comprises isotonic saline solution.
치료학적 용도를 위한 약학적으로 허용되는 담체, 부형제 또는 희석제는 약학 분야에 잘 알려져 있으며, 예를 들어 Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985)에 기술되어 있다.Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).
약학적 담체, 부형제 또는 희석제는 의도한 투여 경로 및 표준 약학 실무에 따라 선택될 수 있다.Pharmaceutical carriers, excipients or diluents may be selected depending on the intended route of administration and standard pharmaceutical practice.
일 구현예에서, 본원에 기술된 약학 조성물은 정맥내, 동맥내, 피하, 진피내 또는 근육내로 투여될 수 있다. 특정 구현예에서, 약학 조성물은 국소 투여 또는 전신 투여를 위해 제형화된다. 전신 투여는 위장관을 통한 흡수를 수반하는 장 투여 또는 비경구 투여를 포함할 수 있다. 본원에 사용된 바와 같이, "비경구 투여"는 정맥내 주사와 같이, 위장관을 통해 이루어지는 것 이외의 다른 임의의 방식으로 투여하는 것을 의미한다. 바람직한 구현예에서, 약학 조성물은 전신 투여용으로 제형화된다. 다른 바람직한 구현예에서, 전신 투여는 정맥내 투여에 의한 것이다. 본 발명의 모든 측면의 일 구현예에서, 본원에서 설명된 FoxM1 변이체, 이의 단편, 또는 이를 엔코딩하는 핵산이 전신으로 투여된다. In one embodiment, the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may include enteral administration or parenteral administration, involving absorption through the gastrointestinal tract. As used herein, “parenteral administration” means administration by any means other than via the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration. In one embodiment of all aspects of the invention, the FoxM1 variant described herein, fragment thereof, or nucleic acid encoding the same is administered systemically.
본원에서 사용된 바와 같이, 용어 "공동-투여"는 여러가지 화합물 또는 조성물을 동일한 환자에게 투여하는 것을 의미한다. 여러가지 화합물 또는 조성물은 동시에, 본질적으로 동시에, 또는 순차적으로 투여될 수 있다.As used herein, the term “co-administration” means administering several compounds or compositions to the same patient. The various compounds or compositions may be administered simultaneously, essentially simultaneously, or sequentially.
본원에 기술된 물질, 조성물 및 방법은 질환, 예를 들어, 항원을 발현하는 질환에 걸린 세포의 존재를 특징으로 하는 질환을 가진 개체를 치료하는 데에 사용될 수 있다. 특히 바람직한 질환은 암 질환이다. 예를 들어, 만약 항원이 바이러스에서 유래되었다면, 물질, 조성물 및 방법은 상기 바이러스에 의해 야기되는 바이러스 질환의 치료에 유용할 수 있다. 만약 항원이 종양 항원이라면, 물질, 조성물 및 방법은 암 질환의 치료에 유용하고, 여기서 암 세포는 상기 종양 항원을 발현한다. The materials, compositions and methods described herein can be used to treat an individual with a disease, e.g., a disease characterized by the presence of diseased cells that express an antigen. A particularly preferred disease is cancer. For example, if the antigen is derived from a virus, the materials, compositions and methods may be useful in the treatment of viral diseases caused by the virus. If the antigen is a tumor antigen, the materials, compositions and methods are useful in the treatment of a cancer disease, wherein cancer cells express the tumor antigen.
본원에 기술된 물질, 조성물 및 방법은 다양한 질환의 치료학적 또는 예방학적 치료에 사용될 수 있고, 여기서 본원에 기술된 바와 같이 면역 효과기 세포의 지원 및/또는 면역 효과기 세포의 활성은 암 및 감염성 질환과 같은 환자에 유리하다. 일 구현예에서, 본원에 기술된 물질, 조성물 및 방법은 항원과 연관된 질환의 예방학적 및/또는 치료학적 치료에 유용하다. The materials, compositions and methods described herein can be used in the therapeutic or prophylactic treatment of a variety of diseases, wherein the support of and/or activation of immune effector cells as described herein is used to treat cancer and infectious diseases. It is advantageous for the same patient. In one embodiment, the materials, compositions and methods described herein are useful for the prophylactic and/or therapeutic treatment of diseases associated with antigens.
용어 "질환"은 개체의 신체에 영향을 미치는 비정상적인 병태를 지칭한다. 질환은 종종 특정 증상 및 신호와 관련있는 의학적인 상태로서 해석된다. 질환은 감염성 질환과 같은 외부 원인으로부터 기원한 인자에 의해 유발될 수 있거나, 또는 자가면역 질환과 같은 내부 기능부전에 의해 유발될 수 있다. 인간에서, "질환"은 보다 넒은 의미에서 개체와 접촉시 질병이 병을 앓고 있는 개체에게 통증, 기능부전, 괴로움, 사회적 문제 또는 사망 또는 비슷한 문제를 유발하는 임의의 병태를 지칭하기 위해 사용된다. 보다 넓은 의미에서, 이는 때때로 상해, 불능, 장애, 증후군, 감염, 단독 증상, 일탈 행위 및 구조적 및 기능적인 비정형성 변형을 포함하며, 다른 맥락 및 다른 목적에서, 이는 구별할 수 있는 범주로 간주될 수 있다. 질환은, 여러가지 질환에 걸려 생활하면 개체의 삶에 대한 관점과 성격을 바꿀 수 있으므로, 일반적으로 개체에게 신체적으로뿐 아니라 감정적으로 영향을 미친다.The term “disease” refers to an abnormal condition affecting an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. The disease may be caused by factors originating from an external source, such as an infectious disease, or may be caused by an internal dysfunction, such as an autoimmune disease. In humans, “disease” is used in a broader sense to refer to any condition that, upon contact with an individual, causes pain, dysfunction, suffering, social problems, death, or similar problems in the afflicted individual. In a broader sense, it sometimes includes injuries, disabilities, disabilities, syndromes, infections, isolated symptoms, aberrant acts and structural and functional atypical deformities, which in other contexts and for other purposes may be considered distinct categories. You can. Diseases generally affect individuals not only physically but also emotionally, as living with various diseases can change an individual's perspective on life and personality.
본 맥락에서, 용어 "치료", "치료하는" 또는 "치료학적 개입"은 질환 또는 장애와 같은 병태를 퇴치할 목적으로 개체를 관리 및 돌보는 것을 의미한다. 이 용어는 증상 또는 합병증을 완화하고/거나, 질환, 장애 또는 병태의 진행을 지연하고/거나, 증상 및 합병증을 완화 또는 경감하고/거나, 질환, 장애 또는 병태를 치유 또는 제거하는 것뿐 아니라 병태를 예방하기 위해, 치료학적으로 효과적인 화합물의 투여와 같이, 개체가 고통받는 소정의 병태에 대한 전 범위 치료를 포함하는 것으로 의도되며, 여기서 예방은 질환, 병태 또는 장애를 퇴치하기 위한 목적으로 개체를 관리 및 돌보는 것으로서 이해될 것이고, 증상 또는 합병증의 개시를 방지하기 위한 활성 화합물의 투여를 포함한다.In this context, the terms “treatment”, “treating” or “therapeutic intervention” mean the management and care of an individual for the purpose of combating a condition such as a disease or disorder. The term refers to alleviating symptoms or complications, delaying the progression of a disease, disorder or condition, alleviating or alleviating symptoms and complications, and/or curing or eliminating a disease, disorder or condition, as well as treating a disease, disorder or condition. Prevention is intended to encompass the full spectrum of treatment for a given condition suffering from an individual, such as the administration of a therapeutically effective compound, wherein prevention refers to treatment of the individual for the purpose of combating the disease, condition or disorder. It will be understood as management and care, and includes the administration of an active compound to prevent the onset of symptoms or complications.
용어 "치료학적 치료"는 개체의 건강 상태를 개선하고/거나 개체의 수명을 연장(증가)하는 임의의 치료를 의미한다. 상기 치료는 개체에서 질환의 소거, 개체에서 질환 진행의 정지 또는 서행, 개체에서 질환 진행의 저해 또는 서행, 개체에서 증상의 빈도 또는 중증도 감소, 및/또는 질환을 현재 앓고 있거나 예전에 걸린 적 있는 개체에서 재발 감소일 수 있다.The term “therapeutic treatment” refers to any treatment that improves the health status of an individual and/or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in the individual, stop or slow the progression of the disease in the individual, inhibit or slow the progression of the disease in the individual, reduce the frequency or severity of symptoms in the individual, and/or treat the individual currently suffering from or previously suffering from the disease. There may be a reduction in recurrence.
용어 "예방학적 치료" 또는 "예방적 치료"는 개체에서 질환이 발생되는 것을 방지하기 위해 의도되는 임의의 치료를 의미한다. 용어 "예방학적 치료" 또는 "예방적 치료"는 본원에서 상호 호환적으로 사용된다.The term “prophylactic treatment” or “prophylactic treatment” refers to any treatment intended to prevent a disease from developing in an individual. The terms “prophylactic treatment” or “prophylactic treatment” are used interchangeably herein.
용어 "개인" 및 "개체"는 본원에서 상호 호환적으로 사용된다. 이들 용어는, 질환 또는 장애 (예를 들어, 암)에 걸릴 수 있거나 또는 취약할 수 있는 인간 또는 기타 포유류 (예, 마우스, 랫, 토끼, 개, 고양이, 소, 돼지, 양, 말 또는 영장류)를 지칭하지만, 질환 또는 장애에 걸렸을 수 있거나 또는 걸리지 않았을 수 있다. 다수 구현예들에서, 개체는 인간이다. 달리 명시되지 않은 한, 용어 "개인" 및 "개체"는 특정 연령을 의미하지 않으며, 따라서 성인, 노인, 어린이 및 신생아를 포괄한다. 본 명세서의 구현예에서, "개체" 또는 "개인"은 "환자"이다.The terms “individual” and “entity” are used interchangeably herein. These terms refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates) that may be susceptible to or susceptible to a disease or disorder (e.g., cancer). However, the person may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms “individual” and “subject” do not refer to a specific age and therefore encompass adults, older adults, children, and newborns. In embodiments herein, “subject” or “individual” is “patient.”
용어 "환자"는 치료가 필요한 개인 또는 개체, 구체적으로 질환에 걸린 개인 또는 개체를 의미한다.The term “patient” refers to an individual or entity in need of treatment, specifically an individual or entity suffering from a disease.
본 명세서의 일 구현예에서, 목표는 종양 항원을 발현하는 암 세포와 같은 항원을 발현하는 질환에 걸린 세포에 대해 면역 반응을 일으키는 것, 종양 세포에 대해 이동성, 침습성, 증식성을 감소시키는 것, 및 종양 항원과 같은 항원을 발현하는 세포를 수반하는 암 질환과 같은 질환을 치료하는 것이다.In one embodiment of the present disclosure, the goal is to mount an immune response against diseased cells expressing the antigen, such as cancer cells expressing tumor antigens, to reduce the migratory, invasive, and proliferative properties of the tumor cells; and treating diseases such as cancer diseases involving cells expressing antigens such as tumor antigens.
본원에서 사용된 바와 같이, "면역 반응"은 항원 또는 항원을 발현하는 세포에 대한 일체화된 신체 반응을 지칭하며, 세포성 면역 반응 및/또는 체액성 면역 반응을 지칭한다. As used herein, “immune response” refers to an integrated body response to an antigen or a cell expressing an antigen, and refers to a cellular and/or humoral immune response.
“세포-매개 면역”, “세포성 면역”, “세포성 면역 반응”, 또는 유사 용어는 항원의 발현을 특징으로 하는, 특히 클래스 I 또는 클래스 II MHC로 항원을 제시하는 것을 특징으로 하는 세포에 대한 세포성 반응을 포함하는 것을 의미한다. 세포성 반응은 “헬퍼” 또는 “살상” 중 하나로서 작동하는 T 세포 또는 T 림프구로 불리는 세포에 관한 것이다. 헬퍼 T 세포 (CD4+ T 세포로도 지칭됨)는 면역 반응을 조절함으로써 중추적인 역할을 하고, 살상 세포 (세포독성 T 세포, 세포용해성 T 세포, CD8+ T 세포 또는 CTL로도 지칭됨)는 암 세포와 같은 질환에 걸린 세포를 죽여 더 많은 질환에 걸린 세포의 발생을 예방한다. “Cell-mediated immunity,” “cellular immunity,” “cellular immune response,” or similar terms refer to cells characterized by the expression of an antigen, particularly by presenting the antigen with class I or class II MHC. This means that it includes a cellular response to The cellular response involves cells called T cells or T lymphocytes that act as either “helpers” or “killers.” Helper T cells (also referred to as CD4+ T cells) play a pivotal role by regulating the immune response, while killer cells (also referred to as cytotoxic T cells, cytolytic T cells, CD8 + T cells, or CTLs) play a central role in controlling the immune response. It kills cells affected by the same disease and prevents the development of more diseased cells.
본 명세서는 보호적, 예방적, 예방학적 및/또는 치료학적일 수 있는 면역 반응을 고려한다. 본원에서 사용된 바와 같이, "면역 반응을 유도하다 [또는 유도하는]"는 유도하기 전 특정 항원에 대한 면역 반응이 없다는 것을 의미할 수 있거나, 또는 유도하기 전 특정 항원에 대한 면역 반응이 기저 수준으로 존재하고 유도 후 강화되는 것을 의미할 수 있다. 따라서, "면역 반응을 유도하다 [또는 유도하는]"는 "면역 반응을 강화하다 [또는 강화하는]"를 포함한다.This specification contemplates immune responses that may be protective, prophylactic, prophylactic and/or therapeutic. As used herein, “induce [or inducing] an immune response” can mean the absence of an immune response to a particular antigen prior to induction, or the absence of an immune response to a particular antigen prior to induction at a basal level. It can mean that it exists and is strengthened after induction. Accordingly, “induce [or induce] an immune response” includes “enhance [or enhance] an immune response.”
용어 "면역요법"은 면역 반응의 유도 또는 강화에 의한 질환 또는 병태의 치료를 의미한다. 용어 "면역요법"은 항원 면역화 또는 항원 백신 접종을 포함한다.The term “immunotherapy” refers to the treatment of a disease or condition by inducing or enhancing an immune response. The term “immunotherapy” includes antigen immunization or antigen vaccination.
용어 "면역화" 또는 "백신 접종"은 예를 들어, 치료학적 또는 예방학적 이유로, 면역 반응을 유도하기 위한 목적으로 항원을 개체에 투여하는 과정을 의미한다.The terms “immunization” or “vaccination” refer to the process of administering an antigen to an individual for the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.
용어 "대식 세포"는 단핵구의 분화에 의해 만들어진 식세포의 하위군을 지칭한다. 염증, 면역 사이토카인 또는 미생물 산물에 의해 활성화된 대식 세포는 비특이적으로 탐식 작용을 수행하여, 수소분해성 및 산화성 공격에 의해 대식 세포 내 외인성 병원체를 사멸시켜 병원체를 분해한다. 분해된 단백질로부터 유래된 펩티드는 대식 세포의 세포 표면 상에 제시되고, 이는 T 세포에 의해 인지될 수 있으며, B 세포 표면 상의 항체와 직접 상호작용하여, T 및 B 세포를 활성화하고 면역 반응을 추가적으로 자극할 수 있다. 대식 세포는 항원 제시 세포 클래스에 속한다. 일 구현예에서, 대식 세포는 비장 대식 세포이다.The term “macrophage” refers to a subgroup of phagocytes created by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines, or microbial products non-specifically perform phagocytosis, killing exogenous pathogens within the macrophages through hydrolytic and oxidative attacks and decomposing the pathogens. Peptides derived from cleaved proteins are presented on the cell surface of macrophages, where they can be recognized by T cells and interact directly with antibodies on the B cell surface, activating T and B cells and further immune responses. It can be stimulating. Macrophages belong to the class of antigen-presenting cells. In one embodiment, the macrophage is a splenic macrophage.
용어 "항원을 수반하는 질환"은 항원이 관련된 임의의 질환, 예를 들어 항원의 존재를 특징으로 하는 질환을 의미한다. 항원을 수반한 질환은 감염성 질환 또는 암 질환 또는 단순 암일 수 있다. 전술한 바와 같이, 항원은 종양-관련 항원, 바이러스 항원 또는 박테리아 항원과 같은 질환-관련 항원일 수 있다. 일 구현예에서, 항원을 수반하는 질환은, 바람직하게는 세포의 표면에, 항원을 발현하는 세포를 수반하는 질환이다. The term “disease involving antigen” refers to any disease in which an antigen is involved, eg, a disease characterized by the presence of an antigen. A disease involving an antigen may be an infectious disease, a cancer disease, or a simple cancer. As mentioned above, the antigen may be a disease-related antigen, such as a tumor-related antigen, a viral antigen, or a bacterial antigen. In one embodiment, the disease involving the antigen is a disease involving cells expressing the antigen, preferably on the surface of the cell.
용어 "암 질환" 또는 "암"은 개체에서 전형적으로 통제되지 않은 세포 증식을 특징으로 하는 병리학적 병태를 지칭하거나 또는 이를 의미한다. 암의 예로는, 비-제한적으로, 암종, 림프종, 모세포종, 육종 및 백혈병을 포함한다. 보다 상세하게는, 이러한 암의 예로는 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 및 뇌하수체 선종을 포함한다. 본 명세서에 따를 때, 용어 "암"은 암 전이를 또한 포함한다.The term “cancer disease” or “cancer” refers to or refers to a pathological condition in an individual typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, Uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, and carcinoma of the sexual and reproductive organs. , Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, Includes spinal tumors, gliomas, meningiomas, and pituitary adenomas. As used herein, the term “cancer” also includes cancer metastases.
암 치료에서 병용 전략 (combination strategy)은, 단일요법 방식의 효과 보다 더 높은 것으로 간주될 수 있는, 달성되는 상승 효과로 인해 적합할 수 있다. 일 구현예에서, 약학 조성물은 면역요법제와 함께 투여된다. 본원에서 사용된 바와 같이, "면역요법제"는 특이적인 면역 반응 및/또는 면역 효과기 기능(들)을 활성화하는데 관여할 수 있는 임의 물질을 의미한다. 본 명세서는 면역요법제로서 항체의 사용을 고려한다. 이론으로 결부시키고자 하는 것은 아니지만, 항체는, 세포자살 유도, 신호 전달 경로의 구성성분의 차단 또는 종양 세포의 증식 저해를 포함하는, 다양한 기전을 통해 암 세포에 대해 치료학적 효과를 달성할 수 있다. 특정 구현예에서, 항체는 단일클론 항체이다. 단일클론 항체는 항체-의존적인 세포 매개 세포독성 (ADCC)을 통해 세포 사멸을 유도할 수 있거나, 보체 단백질에 결합하여, 보체 의존적인 세포독성 (CDC)으로 알려진 직접적인 세포 독성을 유도할 수 있다. 본 발명과 조합하여 사용될 수 있는 항-암 항체 및 잠재적인 항체 표적(괄호 안)에 대한 비-제한적인 예로는, 아바고보맵 (Abagovomab) (CA-125), 압시시맵 (Abciximab) (CD41), 아데카투무맵 (Adecatumumab)(atumumab) (EpCAM), 아푸투주맵 (Afutuzumab) (CD20), 알라시주맵 (Alacizumab pegol) (VEGFR2), 알투모맵 펜테테이트 (Altumomab pentetate) (CEA), 아마투시맵 (Amatuximab) (MORAb-009), 아나투모맵 마페나톡스 (Anatumomab mafenatox) (TAG-72), 아폴리주맵 (Apolizumab) (HLA-DR), 아르시투모맵 (Arcitumomab) (CEA), 아테졸리주맵 (Atezolizumab) (PD-L1), 바비투시맵 (Bavituximab) (포스파티딜세린), 벡투모맵 (Bectumomab) (CD22), 벨리무맵 (Belimumab) (BAFF), 베바시주맵 (Bevacizumab) (VEGF-A), 비바투주맵 메르탄신 (Bivatuzumab mertansine)(CD44 v6), 빌리나투모맵 (Blinatumomab) (CD19), 브렌투시맵 베도틴 (Brentuximab vedotin) (CD30 TNFRSF8), 칸투주맵 메르탄신 (Cantuzumab mertansin) (mucin CanAg), 칸투주맵 라브탄신 (Cantuzumab ravtansine) (MUC1), 카프로맵 펜데티드 (Capromab pendetide) (전립선 암종 세포), 카를루맵 (Carlumab) (CNT0888), 카투맥소맵 (Catumaxomab) (EpCAM, CD3), 세투시맵 (Cetuximab) (EGFR), 시타투주맵 보가톡스 (Citatuzumab bogatox) (EpCAM), 시수투무맵 (Cixutumumab) (IGF-1 수용체), 클라우디시맵 (Claudiximab) (Claudin), 클리바투주맵 테트라세탄 (Clivatuzumab tetraxetan) (MUC1), 코나투무맵 (Conatumumab) (TRAIL-R2), 다세투주맵 (Dacetuzumab) (CD40), 달로투주맵 (Dalotuzumab) (인슐린-유사 성장인자 I 수용체), 데노수맵 (Denosumab) (RANKL), 데투모맵 (Detumomab) (B-림프종 세포), 드로지투맵 (Drozitumab) (DR5), 에크로멕시맵 (Ecromeximab) (GD3 강글리오시드), 에드레콜로맵 (Edrecolomab) (EpCAM), 엘로투주맵 (Elotuzumab) (SLAMF7), 에나바투주맵 (Enavatuzumab) (PDL192), 엔시투시맵 (Ensituximab) (NPC-1C), 에프라투주맵 (Epratuzumab) (CD22), 에르투막소맵 (Ertumaxomab) (HER2/neu, CD3), 에타라시주맵 (Etaracizumab) (인테그린 αγβ3), 파를레투주맵 (Farletuzumab)(폴레이트 수용체 1), FBTA05 (CD20), 피클라투주맵 (Ficlatuzumab) (SCH 900105), 피기투무맵 (Figitumumab) (IGF-1 수용체), 플란보투맵 (Flanvotumab) (당단백질 75), 프레솔리무맵 (Fresolimumab) (TGF-β), 갈리시맵 (Galiximab) (CD80), 가니투맵 (Ganitumab) (IGF-I), 겜투주맵 오조가미신 (Gemtuzumab ozogamicin) (CD33), 게보키주맵 (Gevokizumab) (IL1β), 기렌투시맵 (Girentuximab) (카보닉 안하이드라제 9 (CA-IX)), 글렘바투무맵 베도틴 (Glembatumumab vedotin) (GPNMB), 이브리투모맵 티우세탄 (Ibritumomab tiuxetan) (CD20), 이크루쿠맵 (Icrucumab) (VEGFR-1), 이고보마 (Igovoma) (CA-125), 인다투시맵 라브탄신 (Indatuximab ravtansine) (SDC1), 인테투무맵 (Intetumumab) (CD51), 이노투주맵 오조가미신 (Inotuzumab ozogamicin) (CD22), 이필리무맵 (Ipilimumab) (CD 152), 이라투무맵 (Iratumumab) (CD30), 라베투주맵 (Labetuzumab) (CEA), 렉사투무맵 (Lexatumumab) (TRAIL-R2), 리비비루맵 (Libivirumab) (B형 간염 표면 항원), 린투주맵 (Lintuzumab) (CD33), 로르보투주맵 메르탄신 (Lorvotuzumab mertansine) (CD56), 루카투무맵 (Lucatumumab) (CD40), 루밀리시맵 (Lumiliximab) (CD23), 마파투무맵 (Mapatumumab) (TRAIL-R1), 마투주맵 (Matuzumab) (EGFR), 메폴리주맵 (Mepolizumab) (IL5), 밀라투주맵 (Milatuzumab) (CD74), 미투모맵 (Mitumomab) (GD3 강글리오시드), 모가물리주맵 (Mogamulizumab) (CCR4), 모세투모맵 파수도톡스 (Moxetumomab pasudotox) (CD22), 나콜로맵 타페나톡스 (Nacolomab tafenatox) (C242 항원), 나프투모맵 에스타페나톡스 (Naptumomab estafenatox) (5T4), 나마투맵 (Namatumab) (RON), 넥시투무맵 (Necitumumab) (EGFR), 니모투주맵 (Nimotuzumab) (EGFR), 니볼루맵 (Nivolumab) (IgG4), 요파투무맵 (Ofatumumab) (CD20), 올라라투맵 (Olaratumab) (PDGF-R a), 오나르투주맵 (Onartuzumab) (인간 스캐터 인자 수용체 키나제 (human scatter factor receptor kinase)), 오포르투주맵 모나톡스 (Oportuzumab monatox) (EpCAM), 오레고보맵 (Oregovomab) (CA-125), 옥셀루맵 (Oxelumab) (OX-40), 파니투무맵 (Panitumumab) (EGFR), 파트리투맵 (Patritumab) (HER3), 펨투모마 (Pemtumoma) (MUC1), 페르투주마 (Pertuzuma) (HER2/neu), 핀투모맵 (Pintumomab) (선암종 항원), 프리투무맵 (Pritumumab) (비멘틴 (vimentin)), 라코투모맵 (Racotumomab) (N-글리콜릴뉴라민산), 라드레투맵 (Radretumab) (파이브로넥틴 엑스트라 도메인-B), 라피비루맵 (Rafivirumab) (광견병 바이러스 당단백질), 라무시루맵 (Ramucirumab) (VEGFR2), 릴로투무맵 (Rilotumumab) (HGF), 리투시맵 (Rituximab) (CD20), 로바투무맵 (Robatumumab) (IGF-1 수용체), 살말리주맵 (Samalizumab) (CD200), 시브로투주맵 (Sibrotuzumab) (FAP), 실투시맵 (Siltuximab) (IL6), 타발루맵 (Tabalumab) (BAFF), 타카투주맵 테트라세탄 (Tacatuzumab tetraxetan)(α-페토프로테인), 타플리투모맵 파프톡스 (Taplitumomab paptox) (CD 19), 테나투모맵 (Tenatumomab) (테나신 C), 테프로투무맵 (Teprotumumab) (CD221), 티실리무맵 (Ticilimumab) (CTLA- 4), 티가투주맵 (Tigatuzumab) (TRAIL-R2), TNX-650 (IL13), 토시투모맵 (Tositumomab) (CD20), 트라스투주맵 (Trastuzumab) (HER2/neu), TRBS07 (GD2), 트레멜리무맵 (Tremelimumab) (CTLA-4), 투코투주맵 셀모루킨 (Tucotuzumab celmoleukin) (EpCAM), 우블리투시맵 (Ublituximab) (MS4A1), 우레루맵 (Urelumab) (4-1 BB), 볼로시시맵 (Volociximab) (인테그린 α5β1), 보투무맵 (Votumumab) (종양 항원 CTAA 16.88), 잘루투무맵 (Zalutumumab) (EGFR) 및 자놀리무맵 (Zanolimumab) (CD4)를 포함한다.Combination strategies in cancer treatment may be suitable due to the synergistic effects achieved, which may be considered higher than the effectiveness of monotherapy approaches. In one embodiment, the pharmaceutical composition is administered together with an immunotherapy agent. As used herein, “immunotherapeutic agent” means any agent that can be involved in activating a specific immune response and/or immune effector function(s). This specification contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies may achieve their therapeutic effects on cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting proliferation of tumor cells. . In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death through antibody-dependent cell-mediated cytotoxicity (ADCC), or they can bind to complement proteins and induce direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present invention include Abagovomab (CA-125), Abciximab (CD41) , Adecatumumab (atumumab) (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), Altumomab pentetate (CEA), Amatuximab (Amatuximab) (MORAb-009), Anatumomab mafenatox (TAG-72), Apolizumab (HLA-DR), Arcitumomab (CEA), Atezoli Atezolizumab (PD-L1), Bavituximab (phosphatidylserine), Bectumomab (CD22), Belimumab (BAFF), Bevacizumab (VEGF-A) ), Bivatuzumab mertansine (CD44 v6), Blinatumomab (CD19), Brentuximab vedotin (CD30 TNFRSF8), Cantuzumab mertansin (mucin CanAg), Cantuzumab ravtansine (MUC1), Caproumab pendetide (prostate carcinoma cells), Carlumab (CNT0888), Catumaxomab (EpCAM, CD3) ), Cetuximab (EGFR), Citatuzumab bogatox (EpCAM), Cixutumumab (IGF-1 receptor), Claudiximab (Claudin), Clivatu Clivatuzumab tetraxetan (MUC1), Conatumumab (TRAIL-R2), Dacetuzumab (CD40), Dalotuzumab (insulin-like growth factor I receptor), Denosumab (RANKL), Detumomab (B-lymphoma cells), Drozitumab (DR5), Ecromeximab (GD3 ganglioside), Edrecolomab (Edrecolomab) (EpCAM), Elotuzumab (SLAMF7), Enavatuzumab (PDL192), Ensituximab (NPC-1C), Epratuzumab (CD22), Er Ertumaxomab (HER2/neu, CD3), Etaracizumab (Integrin αγβ3), Farletuzumab (Folate Receptor 1), FBTA05 (CD20), Ficlatuzumab ( SCH 900105), Figitumumab (IGF-1 receptor), Flanvotumab (glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80) , Ganitumab (IGF-I), Gemtuzumab ozogamicin (CD33), Gevokizumab (IL1β), Girentuximab (carbonic anhydrase 9 ( CA-IX)), Glembatumumab vedotin (GPNMB), Ibritumomab tiuxetan (CD20), Icrucumab (VEGFR-1), Igovoma ) (CA-125), Indatuximab ravtansine (SDC1), Intetumumab (CD51), Inotuzumab ozogamicin (CD22), Ipilimumab (CD 152), Iratumumab (CD30), Labetuzumab (CEA), Lexatumumab (TRAIL-R2), Libivirumab (Hepatitis B surface antigen) , Lintuzumab (CD33), Lorvotuzumab mertansine (CD56), Lucatumumab (CD40), Lumiliximab (CD23), Mapatumumab (TRAIL-R1), Matuzumab (EGFR), Mepolizumab (IL5), Milatuzumab (CD74), Mitumomab (GD3 ganglioside), Mogamulizumab (Mogamulizumab) (CCR4), Moxetumomab pasudotox (CD22), Nacolomab tafenatox (C242 antigen), Naptumomab estafenatox (5T4), Namatumab (RON), Necitumumab (EGFR), Nimotuzumab (EGFR), Nivolumab (IgG4), Ofatumumab (CD20), Olaratumumab (Olaratumab) (PDGF-R a), Onartuzumab (human scatter factor receptor kinase), Oportuzumab monatox (EpCAM), oregovomab ( Oregovomab (CA-125), Oxelumab (OX-40), Panitumumab (EGFR), Patritumab (HER3), Pemtumoma (MUC1), Pertuzuma (HER2/neu), Pintumomab (adenocarcinoma antigen), Pritumumab (vimentin), Racotumomab (N-glycolylneuraminic acid) , Radretumab (fibronectin extra domain-B), Rafivirumab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Sibrotuzumab (FAP), Siltuximab (IL6), Tabalumab (BAFF), Tacatuzumab tetraxetan (α-fetoprotein), Taplitumomab paptox (CD 19), Tenatumomab ) (Tenascin C), Teprotumumab (CD221), Ticilimumab (CTLA-4), Tigatuzumab (TRAIL-R2), TNX-650 (IL13), Tositumomab (CD20), Trastuzumab (HER2/neu), TRBS07 (GD2), Tremelimumab (CTLA-4), Tucotuzumab celmoleukin ( EpCAM), Ublituximab (MS4A1), Urelumab (4-1 BB), Volociximab (integrin α5β1), Votumumab (tumor antigen CTAA 16.88), well Includes Zalutumumab (EGFR) and Zanolimumab (CD4).
본 발명의 일 실시예에서는 암의 전이 위험성을 진단 또는 판단하기 위한 정보제공방법을 제공한다. 본 발명에 따른 정보제공 방법은 암에 걸린 대상체로부터 분리된 암세포에서 야생형 FoxM1 단백질 (서열번호 1)에서 Ser25가 인산화되었거나, S25D, S25E 변이를 포함하는 경우, 상기 암에 걸린 대상체가 암 전이 가능성이 높은 것으로 판단하는 단계를 포함하거나, 암에 걸린 대상체로부터 분리된 암세포에서 야생형 FOXM1 유전자 (서열번호 3)에서 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환되어, 이로부터 발현된 단백질의 25번째 아미노산이 Ser에서 Asp, 또는 Glu로 치환된 경우 상기 암에 걸린 대상체가 암 전이 가능성이 높은 것으로 판단하는 단계를 포함한다. One embodiment of the present invention provides a method of providing information for diagnosing or determining the risk of cancer metastasis. The method for providing information according to the present invention is to determine whether Ser25 is phosphorylated in the wild-type FoxM1 protein (SEQ ID NO: 1) or contains S25D or S25E mutations in cancer cells isolated from a subject with cancer, and that the subject with cancer has a possibility of cancer metastasis. 5'-GAT-3', 5'-GAC-3', or the 73rd to 75th nucleic acids in the wild-type FOXM1 gene (SEQ ID NO: 3) in cancer cells isolated from a subject with cancer. , 5'-GAA-3', or 5'-GAG-3', and the 25th amino acid of the protein expressed therefrom is substituted from Ser to Asp, or Glu, the possibility of cancer metastasis in the subject with cancer. This involves determining that it is high.
본 발명의 일 실시예에서, 서열번호 1의 25번째 아미노산 Ser이 Asp, 또는 Glu로 치환된 폴리펩티드를 발현하는 재조합 전이성 암 세포, 및/또는 서열번호 3으로 표시되는 핵산 서열 중 73번째 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환된 핵산을 포함하는 재조합 전이성 암 세포를 제공한다. 5'-GAT-3', 및 5'-GAC-3'는 Asp를, 5'-GAA-3', 및 5'-GAG-3'는 Glu를 코딩하는 코돈이다. 상기 전이성 암 세포는 이동성, 침습성, 및/또는 증식성이 높으므로 전이성 암을 연구하기 위한 세포, 또는 이를 동물 등에 접종하여 전이성 암 in vivo 모델을 제작하는 데에 이용할 수 있다. In one embodiment of the present invention, recombinant metastatic cancer cells expressing a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with Asp or Glu, and/or 73rd to 75th of the nucleic acid sequence represented by SEQ ID NO: 3 Provided are recombinant metastatic cancer cells comprising a nucleic acid wherein the nucleic acid is substituted with 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. 5'-GAT-3', and 5'-GAC-3' are codons that code for Asp, and 5'-GAA-3', and 5'-GAG-3' are codons that code for Glu. Since the metastatic cancer cells have high mobility, invasiveness, and/or proliferative properties, they can be used to study metastatic cancer, or to create an in vivo model of metastatic cancer by inoculating them into animals.
본 발명에서는 FOXM1에 특이적으로 결합하는 shRNA를 포함하는 암 치료용 약학 조성물을 제공한다. The present invention provides a pharmaceutical composition for cancer treatment containing shRNA that specifically binds to FOXM1.
먼저 KEGG 2019 경로 분석을 통해서 폐암세포 A549의 암 전이 조건에서 활성형 PLK1이 발현되는 침습성 세포들의 관련 경로를 분석하였다. p53 신호전달, DNA 복제, 세포 노화, 세포 주기, TGF-β 신호전달 등의 경로들이 관여되었다(도 11A). First, through KEGG 2019 pathway analysis, we analyzed the related pathways of invasive cells expressing active PLK1 under cancer metastasis conditions of lung cancer cell A549. Pathways such as p53 signaling, DNA replication, cell senescence, cell cycle, and TGF-β signaling were involved (Figure 11A).
본 발명의 발명자는 TGF-β처리에 의해 EMT 마커 중 중간엽세포 마커인 N-cadherin, vimentin, SNAI1, SNAI2와 동시적으로 FoxM1의 발현이 증가를 관찰함으로써 암전이 과정에서 FoxM1의 증가가 바이오 마커가 될 수 있음을 확인하였다.The inventors of the present invention observed an increase in the expression of FoxM1 simultaneously with N-cadherin, vimentin, SNAI1, and SNAI2, which are mesenchymal cell markers among EMT markers, by TGF-β treatment, so that the increase in FoxM1 in the process of cancer metastasis is a biomarker. It was confirmed that this can be done.
이를 위하여 TGF-β을 48시간 처리한 비소세포폐암 세포주인 A549, NCI-H358, NCI-H460세포에서 면역블랏법을 이용하여 중간엽세포 마커 (N-cadherin, vimentin, SNAI1, SNAI2)와 함께 FoxM1의 증가가 관찰한 결과 모든 세포에서 일관되게 증가함을 관찰하였다 (도 11B). For this purpose, FoxM1 was identified along with mesenchymal cell markers (N-cadherin, vimentin, SNAI1, SNAI2) using immunoblotting in non-small cell lung cancer cell lines A549, NCI-H358, and NCI-H460 cells treated with TGF-β for 48 hours. As a result of observing the increase, it was observed that it increased consistently in all cells (Figure 11B).
또한 FoxM1의 발현에 의해서 EMT가 변화되는 지 확인하기 위하여, FoxM1을 pLVX-eRFP 벡터에 도입하여 발현시킨 후, A549세포에서 전이인자인 EMT 마커의 변화를 면역블럿법 및 RT-PCR로 관찰하였을 때, FoxM1의 증가와 함께 중간엽세포 마커(E-cadherin, N-cadherin, Vimentin)의 단백질 및 mRNA 발현량을 관찰하였다. E-cadherin 발현은 감소하고 N-cadherin, Vimentin 발현은 증가한 것을 관찰할 수 있었다(도 12A, 12B). In addition, in order to confirm whether EMT is changed by the expression of FoxM1, FoxM1 was introduced into the pLVX-eRFP vector and expressed, and changes in the EMT marker, a metastasis factor, in A549 cells were observed by immunoblotting and RT-PCR. , Protein and mRNA expression levels of mesenchymal cell markers (E-cadherin, N-cadherin, Vimentin) were observed along with an increase in FoxM1. It was observed that E-cadherin expression decreased and N-cadherin and Vimentin expression increased (Figures 12A, 12B).
또한 FoxM1에 의한 암세포의 전이성과 침습성이 증가되는 것을 관찰하기 위하여 transwell insert를 이용한 이동성 실험(migration assay) 및 침습성 실험(invasion assay)을 수행하였다 (도 12C, 12D). Additionally, to observe the increase in metastatic and invasive properties of cancer cells due to FoxM1, migration assays and invasion assays using transwell inserts were performed (Figures 12C, 12D).
FoxM1의 원형(WT) 단백질이 발현되는 A549 세포에서 암세포의 전이성을 관찰하기 위하여 Transwell을 활용한 암세포 이동성 실험을 진행하였다(도 12C). 연구결과 FoxM1의 원형 실험군이 대조군 대비 3배가량 증가되었는데, 이는 양성 대조군인 TGF-β 처리 세포군(5ng/ml)과 같은 양상을 보인다. To observe metastatic properties of cancer cells in A549 cells expressing the original (WT) FoxM1 protein, a cancer cell mobility experiment was performed using Transwell (Figure 12C). As a result of the study, the original experimental group of FoxM1 increased about 3 times compared to the control group, which showed the same pattern as the TGF-β treated cell group (5ng/ml), which was the positive control group.
또한, FoxM1의 원형(WT) 단백질이 발현되는 A549 세포에서의 침습성 촉진 효과를 보기 위해 마트리겔을 이용한 침습성 분석을 이용하여 암세포의 침습성을 관찰하고자 하였다(도 12E). 먼저, 침습능을 평가하기 위하여 마트리겔 insert에 각각의 FoxM1 원형 단백질이 발현하는 세포를 혈청이 없는 배지와 함께 분주하고 실험플레이트에 혈청이 포함된 배지를 분주하여 5일 동안 배양하였다. 침습된 암세포를 크리스탈 바이올렛으로 염색하여 관찰하고 DMSO로 녹인 후 590nm의 파장에서 흡광도를 측정하였다. 그 결과, FoxM1의 원형 단백질을 발현하는 폐암세포군에서 대조군 대비 침습성이 20배가량 증가됨을 관찰하였다. 이는 양성 대조군인 TGF-β 처리 세포군(5ng/ml)과 같은 양상을 보였다. In addition, in order to examine the invasiveness promoting effect in A549 cells expressing the original (WT) protein of FoxM1, we attempted to observe the invasiveness of cancer cells using an invasiveness assay using Matrigel (Figure 12E). First, to evaluate the invasion ability, cells expressing each FoxM1 circular protein were dispensed onto the Matrigel insert with serum-free medium, and serum-containing medium was dispensed onto the experimental plate and cultured for 5 days. Invaded cancer cells were observed by staining with crystal violet, dissolved in DMSO, and absorbance was measured at a wavelength of 590 nm. As a result, it was observed that the invasiveness of the lung cancer cell group expressing the original FoxM1 protein increased about 20-fold compared to the control group. This showed the same pattern as the positive control TGF-β treated cell group (5ng/ml).
그리고 FoxM1의 원형(WT) 단백질이 발현되는 A549 세포에서 면역회피 인자인 CD274 발현을 실시간 중합효소 연쇄반응(Real-time PCR)을 통해 본 결과 FoxM1의 원형(WT) 단백질이 발현되는 A549 세포에서 2.3배 정도 발현이 높음을 관찰할 수 있었다(도 12E). And as a result of examining the expression of CD274, an immune evasion factor, in A549 cells expressing the original (WT) protein of FoxM1 through real-time polymerase chain reaction (Real-time PCR), it was 2.3 in A549 cells expressing the original (WT) protein of FoxM1. It was observed that the expression was about 2-fold higher (Figure 12E).
이를 통해 본 연구자는 FoxM1을 발현시킬 경우 EMT 마커의 증가가 관찰되며 세포의 이동성과 침습성이 TGF-β 처리군과 유사하게 증가함을 알 수 있었다.Through this, the researcher found that when FoxM1 was expressed, an increase in EMT markers was observed, and cell mobility and invasiveness increased similarly to the TGF-β treatment group.
또한 FoxM1에 의한 대식세포의 종양관련 대식세포(TAM)로의 전환에 영향을 미치는 지 관찰하기 위하여 THP-1 세포를 FoxM1을 발현시키는 A549 세포와 공배양을 하여, THP-1세포에서의 종양관련 대식세포 마커의 증가와 분화에 대한 연구를 RT-PCR을 이용하여 수행하였다 (도 13)In addition, in order to observe whether FoxM1 affects the conversion of macrophages into tumor-associated macrophages (TAMs), THP-1 cells were co-cultured with A549 cells expressing FoxM1, and the tumor-related macrophages in THP-1 cells were co-cultured. A study on the increase and differentiation of phagocyte markers was performed using RT-PCR (Figure 13)
FoxM1은 대식세포의 이동 및 분화 등에 관여한다는 연구결과(BALLI, David et al., Oncogene (2012) 31.34:3875-3888; YANG, Yang et al., Diabetes Research and Clinical Practice (2022) 184: 109121)를 기반으로 FoxM1 S25E가 발현되는 암세포가 대식세포 (Macrophage cell)의 분화(polarization)에 영향을 미치는 지 관찰하고자 인간 대식세포인 THP-1 세포와 FoxM1 원형 단백질을 과발현하는 A549 세포를 48시간동안 공배양 후에 M1, M2 마커를 관찰하였다. 그 결과 M1 마커인 INOS, IL12B의 발현은 변화가 없으나 M2 마커인 IL10, CD163, CD206, TGFB1, VEGFA가 모두 FoxM1 원형 단백질이 발현된 세포에서 증가하였다(도 13A). 그중 CD206, TGFB1, VEGFA는 M2d-종양 관련 대식세포(M2d-TAM)의 마커(JAYASINGAM et al., Front. Oncol (2020) 9:1512)로 알려져 있다. FoxM1 원형 단백질이 발현되는 A549 세포와 THP-1을 공배양한 후 A549 세포에서 M2 유도 인자인 IL4, IL6, IL10, VEGFA 그리고 면역회피 인자인 CD274 모두 증가됨을 관찰할 수 있었다. 이는 FoxM1 원형 단백질 발현하는 A549 세포가 THP-1 세포를 M2d-TAM로 분화시킴을 보여준다. Research results show that FoxM1 is involved in the migration and differentiation of macrophages (BALLI, David et al., Oncogene (2012) 31.34:3875-3888; YANG, Yang et al., Diabetes Research and Clinical Practice (2022) 184: 109121) Based on this, to observe whether cancer cells expressing FoxM1 S25E affect polarization of macrophages, THP-1 cells, a human macrophage, and A549 cells overexpressing the FoxM1 circular protein were co-cultured for 48 hours. After culture, M1 and M2 markers were observed. As a result, there was no change in the expression of the M1 markers INOS and IL12B, but the M2 markers IL10, CD163, CD206, TGFB1, and VEGFA all increased in cells expressing the FoxM1 circular protein (Figure 13A). Among them, CD206, TGFB1, and VEGFA are known as markers of M2d-tumor-associated macrophages (M2d-TAM) (JAYASINGAM et al., Front. Oncol (2020) 9:1512). After co-culturing A549 cells expressing the FoxM1 circular protein with THP-1, it was observed that M2-inducing factors IL4, IL6, IL10, VEGFA, and immune evasion factor CD274 were all increased in A549 cells. This shows that A549 cells expressing the FoxM1 circular protein differentiate THP-1 cells into M2d-TAM.
또한 FoxM1 원형 단백질이 발현되는 A549 세포를 48h동안 배양한 배지에서 TGFB1, VEGFA 단백질의 양을 ELISA로 측정한 결과, FoxM1 원형 단백질 발현하는 세포에서 대조군 보다 현저히 증가됨을 관찰하였다(도 13C). 마우스의 폐조직에서 광범위 대식세포 마커인 CD68와 종양관련대식세포 마커인 CD163 마커를 면역염색법으로 분석한 결과, FoxM1 원형(WT) 단백질이 발현된 A549 세포를 주사한 마우스 폐조직에서 CD68, CD163 마커의 발현이 높게 관찰됨을 확인하였다(도 13D). 이를 통해 FoxM1을 발현시킨 암세포를 THP-1 세포와 공동배양시 THP-1의 TAM 마커의 증가 및 분화가 증가됨을 알 수 있다.In addition, as a result of measuring the amount of TGFB1 and VEGFA proteins in the medium in which A549 cells expressing the FoxM1 circular protein were cultured for 48 h by ELISA, it was observed that the amount of TGFB1 and VEGFA proteins was significantly increased in the cells expressing the FoxM1 circular protein compared to the control group (Figure 13C). As a result of immunostaining analysis of CD68, a broad macrophage marker, and CD163, a tumor-related macrophage marker, in mouse lung tissue, CD68 and CD163 markers were found in mouse lung tissue injected with A549 cells expressing FoxM1 circular (WT) protein. It was confirmed that high expression was observed (Figure 13D). This shows that when cancer cells expressing FoxM1 are co-cultured with THP-1 cells, TAM markers and differentiation of THP-1 are increased.
본 발명의 발명자는 FoxM1 mRNA 억제물질인 FoxM1 shRNA에 의한 암세포의 이동성 억제효과를 확인하였다 (도 14).The inventor of the present invention confirmed the inhibitory effect on the mobility of cancer cells by FoxM1 shRNA, a FoxM1 mRNA inhibitor (FIG. 14).
FoxM1의 mRNA 발현을 억제하기 위하여 사람의 FoxM1 mRNA 서열 중 187-207, 709-729 위치의 뉴클레오타이드 서열을 각각 타겟으로 하는 shRNA를 만들고자 pLKO-puro.1 벡터를 이용한 pLKO-puro.1-hFoxM1 플라스미드를 제작하였다. 사람의 FoxM1 shRNA 제작을 위한 타겟 서열로 하기의 서열이 가능하며 shRNA제작을 위한 프라이머로 하기의 서열을 지닌 올리고뉴클레오타이드를 사용할 수 있다. Pubmed에서의 사람의 FoxM1 mRNA 유전자 접근번호는 U74613 이며, 3326bp를 지니고 있다.To suppress the mRNA expression of FoxM1, pLKO-puro.1-hFoxM1 plasmid was created using the pLKO-puro.1 vector to create shRNA targeting the nucleotide sequences at positions 187-207 and 709-729 of the human FoxM1 mRNA sequence. Produced. The following sequences can be used as target sequences for human FoxM1 shRNA production, and oligonucleotides with the following sequences can be used as primers for shRNA production. The accession number for the human FoxM1 mRNA gene in Pubmed is U74613 and has 3326 bp.
# 1 타겟 뉴클레오타이드 위치 187-207#1 Target nucleotide positions 187-207
뉴클레오타이드 서열 nucleotide sequence
sense region: 5'-CATCAGAGGAGGAACCTAAGA-3'sense region: 5'-CATCAGAGGAGGAACCTAAGA-3'
anti-sense region: 5'-TCTTAGGTTCCTCCTCTGATG-3'anti-sense region: 5'-TCTTAGGTTCTCTCCTGATG-3'
shRNA 제작을 위한 프라이머Primers for shRNA production
정방향 프라이머 forward primer
5'-ccgg-CATCAGAGGAGGAACCTAAGA-ctcgag-TCTTAGGTTCCTCCTCTGATG-tttttg-3'5'-ccgg-CATCAGAGGAGGAACCTAAGA-ctcgag-TCTTAGGTTCCTCCTCTGATG-tttttg-3'
역방향 프라이머 reverse primer
5'-aattcaaaaa-CATCAGAGGAGGAACCTAAGA-ctcgag-TCTTAGGTTCCTCCTCTGATG-3'5'-aattcaaaaa-CATCAGAGGAGGAACCTAAGA-ctcgag-TCTTAGGTTCCTCCTCTGATG-3'
# 2 타겟 뉴클레오타이드 위치 709-729#2 Target nucleotide positions 709-729
뉴클레오타이드 서열 nucleotide sequence
sense region: 5'-AGCAAGAGATGGAGGAAAAGG-3'sense region: 5'-AGCAAGAGATGGAGGAAAAGG-3'
anti-sense region: 5'-CCTTTTCCTCCATCTCTTGCT-3'anti-sense region: 5'-CCTTTTCCTCCATCTCTTGCT-3'
shRNA 제작을 위한 프라이머Primers for shRNA production
정방향 프라이머forward primer
5'-ccgg-AGCAAGAGATGGAGGAAAAGG-ctcgag-CCTTTTCCTCCATCTCTTGCT-tttttg-3'5'-ccgg-AGCAAGAGATGGAGGAAAAGG-ctcgag-CCTTTTCCTCCATCTCTTGCT-tttttg-3'
역방향 프라이머reverse primer
5'-aattcaaaaa-AGCAAGAGATGGAGGAAAAGG-ctcgag-CCTTTTCCTCCATCTCTTGCT-3'5'-aattcaaaaa-AGCAAGAGATGGAGGAAAAGG-ctcgag-CCTTTTCCTCCATCTCTTGCT-3'
먼저 제작한 각각의 FoxM1 shRNA 발현용 렌티바이러스를 정제 및 농축시킨 후 제작한 바이러스를 폐암세포인 A549에 감염시켜 FoxM1 발현억제 효과를 확인하고자 mRNA 발현과 단백질 발현 정도를 관찰하였다(도 14A, 14B). 그 결과, 709-729 위치의 뉴클레오타이드 서열을 타겟(타겟 #2, 709-729 bp)으로 하는 shRNA가 FoxM1의 발현억제 효과가 가장 탁월한 것으로 관찰하였다 (도 14A, 14B). First, the lentivirus for expressing FoxM1 shRNA was purified and concentrated, and then the virus was infected with A549 lung cancer cells, and the level of mRNA and protein expression was observed to confirm the effect of suppressing FoxM1 expression (Figures 14A and 14B). . As a result, it was observed that shRNA targeting the nucleotide sequence at position 709-729 (target #2, 709-729 bp) had the most excellent effect on suppressing FoxM1 expression (Figures 14A, 14B).
위의 실험결과로 선정된 FoxM1 shRNA 발현용 렌티바이러스로 암세포의 전이성 억제 효과를 제공한다.Lentivirus for expressing FoxM1 shRNA, selected based on the above experimental results, provides an inhibitory effect on the metastasis of cancer cells.
이를 위하여 제작한 FoxM1 shRNA 발현용 렌티바이러스를 폐암세포인 A549에 감염시켜 폐암세포의 이동성 억제 효과에 대한 실험을 진행하였다(도 14C). For this purpose, the lentivirus for expressing FoxM1 shRNA was infected with lung cancer cells, A549, and an experiment was conducted on the effect of inhibiting the mobility of lung cancer cells (Figure 14C).
제작한 FoxM1 shRNA 발현용 렌티바이러스를 정제 및 농축시킨 후 제작한 바이러스를 폐암세포인 A549에 감염시켜 폐암세포의 전이성에 FoxM1 shRNA가 미치는 영향을 확인하고자 암세포의 이동성 실험(migration assay)을 72시간에 걸쳐 진행하였다(도 14C). 스크래치를 만든 지 72시간 후 암세포의 이동성을 대조군과 비교한 결과, 폐암세포에 FoxM1 shRNA로 FoxM1의 발현을 억제시킨 경우 72시간에 약 -25% 이하로 세포의 상대적 전이성이 감소됨을 관찰하였다(도 14D). After purifying and concentrating the produced lentivirus for expressing FoxM1 shRNA, the produced virus was infected with lung cancer cells, A549, and a cancer cell migration assay was performed at 72 hours to confirm the effect of FoxM1 shRNA on the metastatic properties of lung cancer cells. It progressed over time (Figure 14C). As a result of comparing the mobility of cancer cells with the control group 72 hours after scratching, it was observed that when the expression of FoxM1 was suppressed with FoxM1 shRNA in lung cancer cells, the relative metastatic property of the cells was reduced to about -25% or less in 72 hours (Figure 14D).
TGF-β를 처리에 의해 유도된 암전이가 FoxM1 shRNA (#709) 처리로 FoxM1 mRNA 발현 억제가 간엽 마커(mesenchymal marker)인 CDH2가 현저히 감소됨을 관찰하였다(도 14E).It was observed that the cancer metastasis induced by treatment with TGF-β was suppressed by FoxM1 shRNA (#709) treatment, and that CDH2, a mesenchymal marker, was significantly reduced (FIG. 14E).
FoxM1 shRNA 처리에 의한 FoxM1의 발현 억제로 A549 세포에서 암세포의 세포사멸 정도를 관찰하기 위하여 세포자멸사(아팝토시스)의 대표적인 분해효소인 caspase-3 활성을 측정하기 위하여 caspase-3 assay을 진행하였다(도 14F). 그 결과, FoxM1 shRNA 처리에 의한 FoxM1의 발현 억제로 세포사멸성이 유도 및 증가되는 것을 관찰하였으며 특히 709-729 서열을 표적으로 하는 FoxM1 shRNA는 현저히 caspase-3 활성을 증가시키는 것을 관찰하였다(도 14F).To observe the degree of apoptosis of cancer cells in A549 cells by suppressing the expression of FoxM1 by FoxM1 shRNA treatment, a caspase-3 assay was performed to measure the activity of caspase-3, a representative decomposition enzyme of apoptosis ( Figure 14F). As a result, it was observed that apoptosis was induced and increased by suppressing the expression of FoxM1 by FoxM1 shRNA treatment. In particular, FoxM1 shRNA targeting the 709-729 sequence was observed to significantly increase caspase-3 activity (Figure 14F ).
또한 본 발명은 FoxM1 mRNA 억제물질인 FoxM1 shRNA에 의해, FoxM1 변이체 과발현에 의해 유발되는 종양 미세환경의 단핵세포인 THP-1의 종양관련 대식세포로의 전환을 억제효과를 제공한다 (도 15).In addition, the present invention provides an inhibitory effect on the conversion of THP-1, a mononuclear cell in the tumor microenvironment, into tumor-related macrophages caused by overexpression of a FoxM1 variant by using FoxM1 shRNA, a FoxM1 mRNA suppressor (FIG. 15).
FoxM1 인산화 변이체 과발현 세포에 FoxM1 mRNA 억제물질인 FoxM1 shRNA 감염시킨 결과 FoxM1 발현이 현저히 감소됨을 관찰할 수 있었다(도 15A). 위를 기반으로 상위 세포와 인간 단핵대식세포인 THP-1을 48h동안 공배양 하여 THP-1세포에서 M2 마커인 CD163, CD206, VEGFA 발현을 본 결과, FoxM1 shRNA에 의해 FoxM1 발현이 억제되며 상위 인자들의 발현도 감소됨을 관찰할 수 있었다(도 15B). When cells overexpressing the FoxM1 phosphorylation mutant were infected with FoxM1 shRNA, a FoxM1 mRNA inhibitor, it was observed that FoxM1 expression was significantly reduced (Figure 15A). Based on the above, the parent cells and THP-1, a human mononuclear macrophage, were co-cultured for 48 h and the expression of M2 markers CD163, CD206, and VEGFA in THP-1 cells was observed. As a result, FoxM1 expression was suppressed by FoxM1 shRNA and the upstream factor It was observed that their expression was also reduced (Figure 15B).
또한, FoxM1 인산화 변이체 과발현 세포에 FoxM1 mRNA 억제물질인 FoxM1 shRNA 감염시켜 FoxM1 발현을 억제한 결과 IFITM1의 발현도 따라 감소됨을 관찰할 수 있었다(도 15C).In addition, cells overexpressing the FoxM1 phosphorylation mutant were infected with FoxM1 shRNA, a FoxM1 mRNA inhibitor, to suppress FoxM1 expression, and it was observed that the expression of IFITM1 was also decreased (Figure 15C).
또한 본 발명은 FoxM1 억제제인 thiostepton에 의한 암세포의 전이성 억제 효과를 제공한다.Additionally, the present invention provides an inhibitory effect on the metastasis of cancer cells by thiostepton, a FoxM1 inhibitor.
도 16에서와 같이 FoxM1 억제제인 thiostepton를 이용하여 FoxM1 변이체 과발현에 의해 유발된 폐암세포의 전이성에 미치는 효과를 관찰하고자 약물을 48시간동안 처리하였다.As shown in Figure 16, thiostepton, a FoxM1 inhibitor, was used for 48 hours to observe the effect on metastatic properties of lung cancer cells induced by overexpression of FoxM1 variants.
연구 결과로 thiostepton은 폐암세포 자체의 mesenchymal marker인 CDH2, VIM, SANI1, SNAI2의 발현을 감소시킨 것을 관찰할 수 있었다(도 16A). 또한 thiostepton은 암세포 자체의 전이성뿐만 아니라 종양관련 대식세포 유도인자인 IL6와 VEGFA 발현의 발현을 감소시켰으며 면역회피 인자인 CD274 발현도 감소시킨 것을 관찰할 수 있었다(도 16B). As a result of the study, it was observed that thiostepton decreased the expression of CDH2, VIM, SANI1, and SNAI2, which are mesenchymal markers of lung cancer cells (Figure 16A). In addition, it was observed that thiostepton not only reduced the metastatic properties of the cancer cells themselves, but also reduced the expression of tumor-related macrophage-inducing factors IL6 and VEGFA, and also reduced the expression of CD274, an immune evasion factor (Figure 16B).
아울러, 본 발명은 FoxM1 shRNA 또는 FoxM1억제제를 유효성분으로 포함하는 일차성 암 뿐만 아니라 전이성 암에 대한 항암 치료제를 제공한다.In addition, the present invention provides an anticancer treatment for not only primary cancer but also metastatic cancer, comprising FoxM1 shRNA or FoxM1 inhibitor as an active ingredient.
본 발명의 일 실시예에서, FOXM1 핵산에 의해 발현되는 단백질의 발현 또는 활성을 억제할 수 있는 하나 이상의 억제성 핵산을 포함하는 약학 조성물에 관하여 개시한다. 본 발명의 일 실시예에서, 예를 들어, 환자의 세포와 같은 종양 세포에서 FOXM1 핵산에 의해 코딩되는 단백질의 발현 또는 활성을 억제하기 위한 억제성 핵산을 치료학적 유효량을 투여함으로써 암 환자를 치료할 수 있다. 본 발명의 조성물에 기재된 억제성 핵산은 표적 유전자인 FOXM1의 핵산 서열에 실질적으로 상보적이다. In one embodiment of the present invention, a pharmaceutical composition comprising one or more inhibitory nucleic acids capable of inhibiting the expression or activity of a protein expressed by FOXM1 nucleic acid is disclosed. In one embodiment of the invention, a cancer patient may be treated, for example, by administering a therapeutically effective amount of an inhibitory nucleic acid to inhibit the expression or activity of the protein encoded by the FOXM1 nucleic acid in tumor cells, such as the patient's cells. there is. The inhibitory nucleic acid described in the composition of the present invention is substantially complementary to the nucleic acid sequence of the target gene FOXM1.
본 발명은 암 또는 FOXM1의 발현을 특징으로 하는 질환을 갖는 것으로 진단받은 환자를 치료하는 방법에 관한 것으로, FOXM1 핵산에 의해 코딩되는 단백질의 발현 또는 활성을 억제할 수 있는 억제성 핵산을 투여함으로써 FOXM1의 과발현과 관련된 증상이 완화되어 질환이 치료될 수 있다. The present invention relates to a method of treating a patient diagnosed with cancer or a disease characterized by expression of FOXM1, by administering an inhibitory nucleic acid capable of inhibiting the expression or activity of the protein encoded by the FOXM1 nucleic acid. The symptoms associated with overexpression can be alleviated and the disease can be treated.
본 발명의 일 실시예에 따른 FOXM1의 발현 또는 활성을 억제하는 약제학적 유효량은 당해 기술분야에서 통상의 지식을 가진 자가 환자의 나이, 체중 및 반응 등을 치료 또는 예방될 질환의 맥락에서 고려하여 결정할 수 있다. 본 발명의 일 실시예에서, 유효량은 FOXM1의 발현 또는 활성을 억제할 수 있는 화합물, 약학 조성물, 또는 약물의 조직, 동물, 인간 등에서 암의 억제, 예방 또는 치료를 유발하는 양을 의미한다. The pharmaceutically effective amount for inhibiting the expression or activity of FOXM1 according to an embodiment of the present invention can be determined by a person skilled in the art by considering the patient's age, weight, response, etc. in the context of the disease to be treated or prevented. You can. In one embodiment of the present invention, an effective amount refers to an amount that causes inhibition, prevention or treatment of cancer in tissues, animals, humans, etc. of a compound, pharmaceutical composition, or drug capable of inhibiting the expression or activity of FOXM1.
본 발명의 일 실 시예에서, FOXM1의 발현 또는 활성을 억제할 수 있는 화합물의 약제학적 유효량은 약학 조성물로서 전달될 수 있다. 일 실시예에서, 약학 조성물은 FOXM1의 발현 또는 활성을 억제하는 화합물을 함유하는 제품일 수 있고, 상기 제품은 특정 양의 특정 성분을 포함한 제품 또는 직접적 또는 간접적으로, 특정 양에서 특정 성분의 조합에 의한 제품일 수 있다. In one embodiment of the invention, a pharmaceutically effective amount of a compound capable of inhibiting the expression or activity of FOXM1 can be delivered as a pharmaceutical composition. In one embodiment, the pharmaceutical composition may be a product containing a compound that inhibits the expression or activity of FOXM1, wherein the product contains a specific ingredient in a specific amount or, directly or indirectly, a combination of specific ingredients in a specific amount. It may be a product caused by
일 실시예에서, FOXM1의 발현 또는 활성을 억제하는 억제성 핵산은 FOXM1 핵산의 일부에 결합하여, FOXM1에 의해 코딩되는 단백질의 발현을 조절할 수 있다. 본 발명에 따른 핵산은 표적 영역에 정확히 또는 일부의 불일치 서열을 포함할 수 있다. 다수의 핵산 분자는 FOXM1에 의해 코딩되는 단백질의 발현 또는 활성을 조절할 수 있다. 상기의 핵산 분자는 안티센스 RNA, shRNA, siRNA, miRNA, RNA 압타머, DNA 압타머, 및 decoy RNA를 포함한다. 각각의 핵산 분자는 FOXM1의 발현 또는 활성을 저해하는 데에 사용될 수 있다. In one embodiment, an inhibitory nucleic acid that inhibits the expression or activity of FOXM1 can bind to a portion of the FOXM1 nucleic acid and regulate the expression of the protein encoded by FOXM1. Nucleic acids according to the invention may contain exact or partial mismatch sequences in the target region. A number of nucleic acid molecules can modulate the expression or activity of the protein encoded by FOXM1. The nucleic acid molecules include antisense RNA, shRNA, siRNA, miRNA, RNA aptamer, DNA aptamer, and decoy RNA. Each nucleic acid molecule can be used to inhibit the expression or activity of FOXM1.
일 실시예에서, 억제성 핵산은 약 15 내지 약 50개의 염기쌍, 예를 들어 21 내지 25개의 염기쌍을 포함하고, 세포 내 표적 유전자 또는 RNA와 동일하거나 거의 동일한 핵산 서열을 갖는 이중 가닥 구조를 포함하는 siRNA일 수 있다. 안티센스 핵산은 모폴리노(morpholinos), 2′-O-메틸 폴리뉴클레오티드, DNA, 또는 RNA 등을 포함하나, 이에 제한되는 것은 아니다. RNA 폴리머라아제 III 전사된 DNA는 U6 프로모터와 같은 프로모터를 함유한다. 이들 DNA는 전사되어 세포 내에서 siRNA로서 작동할 수 있는 작은 헤어핀 RNA 또는 안티센스 RNA로 작동할 수 있는 선형 RNA를 생성한다. FOXM1의 발현 또는 활성을 억제하는 억제성 핵산은 리보뉴클레오티드, 디옥시리보뉴클레오티드, 합성 뉴클레오티드 또는 표적 RNA 및/또는 유전자가 저해될 수 있는 적합한 조합을 포함할 수 있다. 또한, 핵산의 형태는 단일 가닥, 이중 가닥, 삼중 가닥 또는 사중 가닥일 수 있다. In one embodiment, the inhibitory nucleic acid comprises a double-stranded structure comprising about 15 to about 50 base pairs, for example 21 to 25 base pairs, and having a nucleic acid sequence that is identical or nearly identical to the target gene or RNA in the cell. It may be siRNA. Antisense nucleic acids include, but are not limited to, morpholinos, 2'-O-methyl polynucleotides, DNA, or RNA. RNA polymerase III transcribed DNA contains promoters such as the U6 promoter. These DNAs are transcribed to produce linear RNAs that can act as small hairpin RNAs or antisense RNAs that can act as siRNAs within cells. Inhibitory nucleic acids that inhibit the expression or activity of FOXM1 may include ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. Additionally, the form of the nucleic acid may be single-stranded, double-stranded, triple-stranded, or quadruple-stranded.
억제성 핵산은 화학적 또는 생물학적으로 생산될 수 있거나 플라스미드 또는 재조합 바이러스 벡터에 의해 발현될 수 있다. Inhibitory nucleic acids can be produced chemically or biologically or expressed by plasmids or recombinant viral vectors.
본 발명에 따른 억제성 핵산은 짧은 헤어핀 RNA (shRNA)일 수 있다. shRNA는 생체 외에서 합성되거나 in vivo에서 RNA 폴리머라아제 III 프로모터로부터 전사되어 형성될 수 있다. 이러한 shRNA를 제조하고 포유동물 세포에서 유전자 절단하는 데에 사용하는 것은 Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., Conklin, D. S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 16:948-58; McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., Kay M. A. (2002). RNA interference in adult mice. Nature, 418:38-9; McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., Sharp P. A. (2002). Gene silencing using micro-RNA designed hairpins. RNA, 8:842-50; Yu, J.-Y., DeRuiter, S. L., Turner, D. L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA, 99:6047-52 등에 기술되어 있다. 이러한 shRNA는 세포 내에서 또는 동물 내에서 조작되어 요망되는 유전자를 계속적이고 안정적으로 억제할 수 있다. 통상의 기술자는 세포 내에서 shRNA가 처리되어 siRNA가 생성될 수 있음을 이해할 것이다. Inhibitory nucleic acids according to the invention may be short hairpin RNA (shRNA). shRNA can be synthesized in vitro or formed in vivo by transcription from an RNA polymerase III promoter. The preparation of these shRNAs and their use for gene cleavage in mammalian cells is described by Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 16:948-58; McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., Kay M. A. (2002). RNA interference in adult mice. Nature, 418:38-9; McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., Sharp P. A. (2002). Gene silencing using micro-RNA designed hairpins. RNA, 8:842-50; Yu, J.-Y., DeRuiter, S. L., Turner, D. L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA, 99:6047-52, etc. These shRNAs can be manipulated within cells or within animals to continuously and stably suppress the desired gene. Those skilled in the art will understand that shRNA can be processed within cells to produce siRNA.
일 실시예에서, shRNA는 서열번호 7 내지 10 중 어느 하나의 서열을 포함한다. 일 실시예에서 shRNA는 서열번호 7 또는 8의 서열을 포함하고, 이는 생체 내에서 FOXM1의 발현 또는 활성을 억제하는 데에 사용될 수 있다. 일 실시예에서, shRNA는 서열번호 9 또는 10의 서열을 포함하고, 이는 생체 내에서 FOXM1의 발현 또는 활성을 억제하는 데에 사용될 수 있다.In one embodiment, the shRNA comprises the sequence of any one of SEQ ID NOs: 7-10. In one embodiment, the shRNA includes the sequence of SEQ ID NO: 7 or 8, which can be used to inhibit the expression or activity of FOXM1 in vivo. In one embodiment, the shRNA comprises the sequence of SEQ ID NO: 9 or 10, which can be used to inhibit the expression or activity of FOXM1 in vivo.
상기의 억제성 핵산은 15개 내지 30개의 뉴클레오티드 길이의 상보성 영역 사이에 비상보성 영역(3 내지 6개의 뉴클레오타이드 길이)을 포함하여 표적 RNA와 듀플렉스를 형성하도록 설계되고 합성될 수 있다. FOXM1에 의해 코딩되는 핵산의 발현 또는 활성을 억제할 수 있는 억제성 핵산은 18 내지 100개의 뉴클레오티드 길이일 수 있고, 일 실시예에서 통상의 기술자는 성숙한 형태로 변형되도록 설계할 수 있다. 예를 들어, 성숙된 miRNA는 19 내지 30개 뉴클레오티드, 21 내지 25개의 뉴클레오티드, 또는 21, 22, 23, 24, 또는 25개의 뉴클레오티드 길이일 수 있고, 여기서 miRNA 전구체는 70 내지 100개의 뉴클레오티드 길이를 갖고 헤어핀 구조를 가질 수 있다. The inhibitory nucleic acid can be designed and synthesized to form a duplex with the target RNA, including a non-complementary region (3 to 6 nucleotides long) between the complementary regions of 15 to 30 nucleotides in length. The inhibitory nucleic acid capable of inhibiting the expression or activity of the nucleic acid encoded by FOXM1 can be 18 to 100 nucleotides in length, and in one embodiment, can be designed by one of ordinary skill in the art to be modified into the mature form. For example, the mature miRNA may be 19 to 30 nucleotides, 21 to 25 nucleotides, or 21, 22, 23, 24, or 25 nucleotides in length, wherein the miRNA precursor is 70 to 100 nucleotides in length. It may have a hairpin structure.
상술된 억제성 핵산은 RNA, DNA 또는 이 둘의 올리고머 또는 폴리머일 수 있다. 이 용어는 자연적으로 발생되는 핵염기, 당, 및 백본뿐 아니라 기능적 유사성을 갖는 비-자연적 부분을 갖는 올리고뉴클레오티드를 포함할 수 있다. The inhibitory nucleic acids described above may be RNA, DNA, or oligomers or polymers of both. The term can include oligonucleotides having naturally occurring nucleobases, sugars, and backbones as well as non-natural moieties with functional similarities.
억제성 핵산은 FOXM1에 결합될 수 있을 정도로 충분한 상보적 뉴클레오티드 서열을 포함할 수 있다. 일 실시예에서, 충분한 상보성은 12 내지 25개의 뉴클레오티드, 13 내지 23개의 뉴클레오티드, 14 내지 23개의 뉴클레오티드, 또는 15 내지 23개의 뉴클레오티드일 수 있다. 본 발명의 핵산 분자는 사실 임의의 길이일 수 있다. FOXM1을 코딩하는 단백질의 발현 또는 활성을 억제할 수 있는 억제성 핵산은 20 내지 100개의 뉴클레오티드 길이, 예를 들어, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 또는 99 개의 뉴클레오티드 길이일 수 있다. The inhibitory nucleic acid may comprise sufficient complementary nucleotide sequence to bind to FOXM1. In one embodiment, sufficient complementarity may be 12 to 25 nucleotides, 13 to 23 nucleotides, 14 to 23 nucleotides, or 15 to 23 nucleotides. The nucleic acid molecules of the invention may be of virtually any length. Inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoding FOXM1 are 20 to 100 nucleotides long, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. , 32, 33, 34, 35, 36, 37 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, It may be 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 nucleotides in length.
일 실시예에서, 억제성 핵산은 만약 본 발명의 억제성 핵산의 존재 하에서 표적 유전자 전사체의 안정성이 부재 시의 안정성보다 감소되고/거나 억제성 핵산이 적어도 17개, 적어도 18개, 적어도 19개, 적어도 20개, 적어도 21개, 적어도 22개, 또는 적어도 23개의 길이에 대하여 표적 전사체와 90% 이상의 상동성있는 서열을 갖고/거나, 엄격한 조건 하에서 억제성 핵산이 표적 전사체에 결합한다면 FOXM1을 표적으로 하는 것으로 간주된다. In one embodiment, the inhibitory nucleic acid is selected from the group consisting of at least 17, at least 18, and at least 19 inhibitory nucleic acids if the stability of the target gene transcript in the presence of the inhibitory nucleic acid of the invention is reduced compared to stability in the absence of the inhibitory nucleic acid. , at least 20, at least 21, at least 22, or at least 23 have a sequence that is at least 90% homologous to the target transcript for a length of at least 20, at least 21, at least 22, or at least 23, and/or FOXM1 if the inhibitory nucleic acid binds to the target transcript under stringent conditions. is considered to be targeting.
억제성 핵산은 발현 벡터를 이용하여 생물학적으로 생산될 수 있다. Inhibitory nucleic acids can be produced biologically using expression vectors.
본 발명의 억제성 핵산은 in vivo에서 세포 기반으로 합성되거나 in vitro에서 화학적 합성을 통해 생산될 수 있다. FOXM1에 의해 코딩되는 단백질의 발현 또는 활성을 억제하는 억제성 핵산은 당해 기술 분야의 공지된 방법에 따라 화학적 합성 및/또는 효소적 라이게이션 반응을 사용하여 생산될 수 있다. The inhibitory nucleic acid of the present invention can be synthesized in vivo on a cell basis or produced in vitro through chemical synthesis. Inhibitory nucleic acids that inhibit the expression or activity of the protein encoded by FOXM1 can be produced using chemical synthesis and/or enzymatic ligation reactions according to methods known in the art.
상기의 억제성 핵산은 표적 핵산에 충분히 상보적인 영역을 포함하고, miRNA가 표적 핵산과 이중 구조를 형성할 정도의 충분한 길이로 구성된다. FOXM1에 의해 코딩되는 단백질의 발현 또는 활성을 억제할 수 있는 억제성 핵산은 표적 RNA에 대하여 부분 적으로, 또는 완전히 상보적인 영역을 포함한다. FOXM1에 의해 코딩되는 단백질의 발현 또는 활성을 억제할 수 있는 억제성 핵산과 표적 서열이 안벽하게 상보적일 필요는 없지만, 표적 유전자의 발현을 억제할 정도로는 상응해야 한다. The inhibitory nucleic acid contains a region sufficiently complementary to the target nucleic acid and is of sufficient length so that the miRNA forms a duplex structure with the target nucleic acid. Inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoded by FOXM1 include a region that is partially or fully complementary to the target RNA. The inhibitory nucleic acid capable of inhibiting the expression or activity of the protein encoded by FOXM1 and the target sequence do not have to be completely complementary, but must be compatible enough to inhibit expression of the target gene.
억제성 핵산은 요망되는 특성을 갖도록 변형되어 합성될 수 있다. 예를 들어, 변형은 안정성을 향상시키거나, 표적 핵산과 열역학적으로 하이브리드 확률을 높이거나 특정 조직 또는 세포 유형에 대한 표적성을 높이거나, 세포에 대한 침투성을 높이도록 수행될 수 있다. 변형은 서열 특이성을 높이고 비특이적 결합을 줄이기 위하여 수행될 수 있다. Inhibitory nucleic acids can be synthesized and modified to have the desired properties. For example, modifications may be made to improve stability, increase the probability of thermodynamic hybridization with the target nucleic acid, increase targetability to a specific tissue or cell type, or increase permeability to cells. Modifications may be performed to increase sequence specificity and reduce non-specific binding.
억제성 핵산 분자는 FOXM1을 코딩하는 핵산의 일부와 실질적으로 동일한 뉴클레오티드 서열을 가질 수 있다. 상기에 기재된 바와 같이, FOXM1을 코딩하는 핵산의 적어도 일부와 실질적으로 동일한 단일가닥 올리고뉴클레오티드는 암을 갖거나 암을 가질 위험이 있는 환자에 투여될 수 있다. The inhibitory nucleic acid molecule may have a nucleotide sequence that is substantially identical to a portion of the nucleic acid encoding FOXM1. As described above, single-stranded oligonucleotides that are substantially identical to at least a portion of the nucleic acid encoding FOXM1 can be administered to patients who have cancer or are at risk of having cancer.
억제성 핵산은 네이키드 플라스미드 또는 아데노바이러스, 렌티바이러스, 알파바이러스, 및 아데노-관련 바이러스를 포함하는 DNA 바이러스와 RNA 바이러스를 포함하는 바이러스 벡터를 포함하는 발현 벡터나 리포좀에 제형화된 다른 DNA를 포함하는 다양한 임의의 형태로 세포로 전달될 수 있다. 필요한 핵산의 양은 당해 기술 분야의 통상의 기술자가 전달 제형이나 사용하는 핵산이 DNA인지 RNA인지에 따라 결정할 수 있을 것이다. Inhibitory nucleic acids include naked plasmids or other DNA formulated in liposomes or expression vectors, including viral vectors, including DNA viruses and RNA viruses, including adenoviruses, lentiviruses, alphaviruses, and adeno-associated viruses. It can be delivered to cells in any of a variety of forms. The amount of nucleic acid needed can be determined by a person skilled in the art depending on the delivery formulation and whether the nucleic acid used is DNA or RNA.
상기의 FOXM1을 코딩하는 단백질의 발현 또는 활성을 억제할 수 있는 억제성 핵산은 DNA 또는 RNA 벡터에 삽입된 전사 유닛으로부터 발현될 수 있다. 재조합 벡터는 DNA 플라스미드 도는 바이러스 벡터일 수 있다. FOXM1을 코딩하는 단백질의 발현 또는 활성을 억제할 수 있는 억제성 핵산을 생산하기에 적합한 바이러스 벡터는, 예를 들어, 아데노-관련 바이러스, 레트로바이러스, 아데노바이러스, 또는 알파바이러스에 기반하여 설계될 수 있으나, 이에 제한되는 것은 아니다. FOXM1을 코딩하는 단백질의 발현 또는 활성을 억제할 수 있는 억제성 핵산을 발현할 수 있는 재조합 벡터는 상기의 방법에 따라 전달될 수 있고, 표적 세포에서 지속될 수 있거나 핵산 분자의 일시적 발현만을 제공할 수 있다. 이러한 벡터는 필요에 따라 반복적으로 투여될 수 있다. 일단 발현되면 억제성 핵산은 표적 RNA와 상호작용하여 miRNA 활성을 억제한다. 파포바바이러스를 포함하여, 예를 들어, SV40, 아데노바이러스, 백시니아 바이러스, 아데노-관련 바이러스, 헤르페스바이러스, 및 조류, 설치류, 및 인간 기원 레트로바이러스와 같은 수 많은 바이러스가 본 발명에 이용될 수 있다. 일 실시예에서, 렌티바이러스 벡터도 본 발명에 사용될 수 있다. 일 실시예에서, 렌티바이러스 벡터는 FOXM1에 대한 하나 이상의 shRNA를 발현하도록 조작된 독시사이클린-유도가능한 렌티바이러스 벡터일 수 있다. The inhibitory nucleic acid capable of inhibiting the expression or activity of the protein encoding FOXM1 may be expressed from a transcription unit inserted into a DNA or RNA vector. Recombinant vectors may be DNA plasmids or viral vectors. Viral vectors suitable for producing inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoding FOXM1 can be designed, for example, based on adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. However, it is not limited to this. Recombinant vectors capable of expressing inhibitory nucleic acids capable of inhibiting the expression or activity of the protein encoding FOXM1 can be delivered according to the above methods and can persist in target cells or provide only transient expression of the nucleic acid molecule. there is. These vectors can be administered repeatedly as needed. Once expressed, the inhibitory nucleic acid interacts with the target RNA and inhibits miRNA activity. Numerous viruses can be used in the present invention, including papovaviruses, such as SV40, adenovirus, vaccinia virus, adeno-associated virus, herpesvirus, and retroviruses of avian, rodent, and human origin. there is. In one embodiment, lentiviral vectors may also be used in the present invention. In one embodiment, the lentiviral vector may be a doxycycline-inducible lentiviral vector engineered to express one or more shRNAs against FOXM1.
억제성 핵산을 발현하는 벡터의 전달은 혈관내 또는 근육내 주사와 같이 전신으로, 또는 표적 기관이나 조직 등으로 국소적으로 투여될 수 있다. Delivery of a vector expressing an inhibitory nucleic acid can be administered systemically, such as by intravascular or intramuscular injection, or locally to a target organ or tissue.
DNA 내 포스포다이에스터 결합을 자르는 핵산 가수분해 효소는 대부분의 세포에서 발현되기 때문에, 억제성 올리고뉴클레오티드와 같은 변형되지 않은 DNA는 대부분 분해되지 않기 위해 일반적으로 변형이 일어난다. 또한, 안티센스의 대부분의 표적은 세포 내에 존재하기 때문에, 핵산이 세포 내로 들어가는 것을 고려해야 한다. 임상적 사용을 위해 분해되지 않도록 변형된 뉴클레오티드를 갖는 억제성 핵산이 선호된다. 또한, 특정 세포를 표적으로 할 수 있는 선택성을 높이거나 세포 막 등을 통과할 수 있도록 하기 위하여 다른 분자가 융합되기도 한다. Since nucleic acid hydrolases that cleave phosphodiester bonds in DNA are expressed in most cells, unmodified DNA, such as inhibitory oligonucleotides, is usually modified to avoid degradation. Additionally, since most targets of antisense exist within cells, the entry of nucleic acids into cells must be considered. For clinical use, inhibitory nucleic acids with nucleotides modified to prevent degradation are preferred. In addition, other molecules may be fused to increase selectivity for targeting specific cells or to enable them to pass through cell membranes.
억제성 핵산의 화학적 변형을 통해 생리학적 활성을 현저히 향상시킬 수 있다. 안티센스핵산을 포스포로시오에이트 변형함으로써 세포 표면 단백질에 대한 결합력을 향상시킬 수 있다. PMO-변형된 안티센스 핵산에 대하여 양전하를 띄는 아르기닌이 풍부한 펩티드를 융합시킴으로써 세포 전달을 개선할 수 있다. Physiological activity can be significantly improved through chemical modification of inhibitory nucleic acids. Antisense nucleic acids can be modified with phosphorothioate to improve binding ability to cell surface proteins. Cell delivery can be improved by fusing positively charged arginine-rich peptides to PMO-modified antisense nucleic acids.
세포내 전달 시스템은 안티센스 핵산 융합체와 양이온성 지질 전달체, 세포 특이적 수용체에 결합하는 운반체 분자, 사이클로덱스트린, 덴드리머, 미세입자, 및 거대분자를 포함한다. 이 전달 시스템은 안티센스 핵산을 핵산 분해로부터 보호하고/거나 흡수성 엔도사이토시스를 촉진하여 세포내 전달 효율을 향상시킬 수 있다. Intracellular delivery systems include antisense nucleic acid fusions and cationic lipid carriers, carrier molecules that bind to cell-specific receptors, cyclodextrins, dendrimers, microparticles, and macromolecules. This delivery system can improve intracellular delivery efficiency by protecting antisense nucleic acids from nucleic acid degradation and/or promoting absorptive endocytosis.
거대분자에는 세포-관통 펩티드 (CPP), 이황화 결합을 통해 안티센스 핵산에 융합된 양이온성 짧은 펩티드 서열을 포함한다. 일반적으로 사용되는 CPP는 penetratin, HIV TAT 펩티드 48-60, 및 transportan을 포함한다. 또한, 리포솜 전달 시스템에 다이올레일포스파티딜에탄올아민을 결합함으로써 엔도좀 막을 불안정화시키고 엔도사이토시스 후에 안티센스 핵산의 방출을 촉진할 수 있다. Macromolecules include cell-penetrating peptides (CPPs), short cationic peptide sequences fused to antisense nucleic acids through disulfide bonds. Commonly used CPPs include penetratin, HIV TAT peptide 48-60, and transportan. Additionally, by binding dioleylphosphatidylethanolamine to the liposome delivery system, it is possible to destabilize the endosomal membrane and promote the release of antisense nucleic acids after endocytosis.
투여 후 생리학적 활성을 증진시키기 위해서 불활성 생분해성 알부민 폴리머 매트릭스에 캡슐화할 수 있는데, 이를 통해 9% 내지 70%의 생리학적 활성을 증진시킬 수 있는 것으로 보고되었다. 또한, 반감기나 분포용적과 같은 약동학적 지표 역시 미세캡슐화를 통해 증가시킬 수 있음이 보고된 바 있다.To enhance physiological activity after administration, it can be encapsulated in an inert, biodegradable albumin polymer matrix, and it has been reported that this can improve physiological activity by 9% to 70%. Additionally, it has been reported that pharmacokinetic indices such as half-life and volume of distribution can also be increased through microencapsulation.
몇몇 나노입자 기반의 siRNA 전달 시스템이 FDA에 승인되어 암 치료 관련 임상에 도입된 바 있다. 현재 암 치료에 관하여 임상 시험 중인 모든 나노입자로 제형화된 siRNA 전달 시스템은 폴리머 또는 리포좀에 기반한다. Several nanoparticle-based siRNA delivery systems have been approved by the FDA and have been introduced into clinical trials for cancer treatment. All nanoparticle-formulated siRNA delivery systems currently in clinical trials for cancer treatment are based on polymers or liposomes.
효과적인 siRNA 전달을 위하여 표적 리간드에 융합된 나노입자는 종양 표면 수용체에 결합할 수 있는 확률이 증가하지만, 이러한 공정으로 인해 나노입자의 전체적인 크기가 증가하게 된다. 나노입자를 PEG로 코팅하면 RES에 의한 흡수를 감소시키고, 결과적으로 순환 반감기를 증가시키지만, PEG 분자가 공간학적으로 선택적 결합을 방해하기 때문에 표적 특이성이 감소하게 된다. 따라서, 적절한 세포 특이적 표적 모이어티를 선택하고 안정하고 효능이 있는 나노입자 전달 시스템을 설계하는 것이 중요하다. 양이온성 지질, 폴리머, 덴드리머, 및 무기 나노입자와 같은 다양한 나노입자 기반의 전달 시스템이 in vitro 및 in vivo에서 siRNA를 효과적으로 전달할 수 있는 것으로 공지되어 있다. For effective siRNA delivery, nanoparticles fused to targeting ligands have an increased probability of binding to tumor surface receptors, but this process also increases the overall size of the nanoparticles. Coating the nanoparticles with PEG reduces uptake by RES and consequently increases the circulating half-life, but reduces target specificity because the PEG molecules interfere with spatially selective binding. Therefore, it is important to select appropriate cell-specific targeting moieties and design stable and efficacious nanoparticle delivery systems. It is known that various nanoparticle-based delivery systems, such as cationic lipids, polymers, dendrimers, and inorganic nanoparticles, can effectively deliver siRNA in vitro and in vivo.
안티센스 핵산은 흡수가 가능하도록 화학적인 변형을 수반하여 식염수 내에 제형화되어 경구 투여와 같이 전신으로, 또는 종양으로의 국소 투입을 통해 전달될 수 있다. 이들의 포스포로시오에이트 백본은 혈청 단백질에 결합하여 신장에 의한 분비를 늦춘다. 방향성 핵염기는 혈청 내 및 세포 표면 상의 다른 소수성 분자와 상호작용한다. 많은 형태의 세포들이 in vivo에서 올리고뉴클레오티드를 흡수하는 표면 수용체를 발현되는데, 배양된 세포에서는 종종 사라지기도 하며, 이는 in vivo에서보다 배양 시 지질이 ASO (allele-specific oligonucleotide) 전달에 더 중요하게 보이는지를 설명한다. Antisense nucleic acids can be formulated in saline solution with chemical modifications to enable absorption and delivered systemically, such as by oral administration, or through topical administration to tumors. Their phosphorothiate backbone binds to serum proteins and slows secretion by the kidneys. The aromatic nucleobases interact with other hydrophobic molecules in serum and on the cell surface. Many cell types express surface receptors that take up oligonucleotides in vivo, but these often disappear in cultured cells, suggesting that lipids may be more important for allele-specific oligonucleotide (ASO) delivery in culture than in vivo. Explain.
전달은 단일 가닥 핵산보다는 이중 가닥 RNA가 더 어렵다. siRNA에서, 모든 방향족 핵염기는 안쪽에 위치하고, 오직 상당량 수화된 인산만이 이중 가닥의 바깥쪽에 위치한다. 수화된 표면은 세포 표면과는 거의 상호작용하지 못하고, 소변으로 빠르게 배출된다. 따라서 연구자들은 siRNA의 전달체를 개발하기 위해 많은 연구를 수행하였다. siRNA를 전달하기 위한 주된 기술은, 비록 요망되는 결과가 펩티드 형질도입 도메인 및 양이온성 폴리머로도 얻어지긴 하였으나, RNA를 양이온성 및 중성 지질과 혼합하는 것이다. 제제 내에 PEG화된 지질을 포함함으로써, 입자의 순환 반감기를 연장시킬 수 있다. siRNA의 하나의 가닥에 콜레스테롤을 융합함으로써 마우스의 간에서 효과적으로 녹다운을 유발하였으나 유효성분의 필요량이 통상의 지질 기반 제형보다 수배 높에 낮다는 단점이 있었다. Delivery of double-stranded RNA is more difficult than single-stranded nucleic acid. In siRNA, all aromatic nucleobases are located on the inside, and only the significantly hydrated phosphates are located on the outside of the double strand. The hydrated surface has little interaction with the cell surface and is quickly excreted in the urine. Therefore, researchers have conducted a lot of research to develop siRNA delivery vehicles. The main technique for delivering siRNA is mixing RNA with cationic and neutral lipids, although desired results have also been obtained with peptide transduction domains and cationic polymers. By including PEGylated lipids in the formulation, the circulating half-life of the particles can be extended. By fusing cholesterol to one strand of siRNA, it effectively induced knockdown in the liver of mice, but it had the disadvantage of being that the amount of active ingredient required was several times higher than that of conventional lipid-based formulations.
단일 가닥 DNA 또는 RNA의 최적화 중 한가지 방법은 포스포다이에스터 결합 위치에 포스포로시오에이트 (PS) 결합을 도입하는 것과 같이 핵산 분해 효소에 대한 저항성을 증가시키기 위한 화학적 변형을 사용하는 것이다. 이와 같은 변형은 핵산 분해 효소에 의한 소화에 대한 안정성을 크게 향상시킨다. PS 결합은 또한 in vivo에서 혈청 단백질에 대한 결합력을 향상시키고, 반감지를 증가시키며 조직으로의 활성 성분이 더 잘 전달될 수 있도록 한다. PS 변형만을 포함하는 ASO는 안티센스 효과를 세포 내에서 만들어낼 수 있으나 효력이 항상 높지 않을 뿐더러 결과가 신뢰할만큼 일상적이지도 않다. One way to optimize single-stranded DNA or RNA is to use chemical modifications to increase resistance to nucleases, such as introducing phosphorothioate (PS) bonds at the phosphodiester bond sites. Such modifications greatly improve stability against digestion by nucleolytic enzymes. PS binding also improves binding to serum proteins in vivo, increases half-sensitivity, and allows better delivery of the active ingredient to tissues. ASOs containing only PS modifications can produce an antisense effect intracellularly, but the potency is not always high and the results are not routine enough to be reliable.
화학적 변형은 또한 상보적 서열에 대한 핵산의 결합 친화도를 높임으로써 효능 및 선택성을 증진시킬 수 있다. 널리 사용되는 변형은 2′-O-methyl (2′-O-Me), 2′-fluoro (2′-F), 및 2′-O-methoxyethyl (2′-MOE) RNA이다. 또한 라이보오스의 2' 및 4' 위치 사이의 메틸렌 브릿지를 함유하는 LNA (locked nucleic acid)로 올리고뉴클레오티드를 변형함으로써 더 높은 친화력을 얻을 수 있다. 브릿지는 상보적 서열에 대한 결합 및 높은 친화력을 달성할 수 있는 이상적인 구조로 라이보오스 고리를 고정시킨다. 관련된 BNA(bridged nucleic acid) 화합물이 개발되었고 이러한 요망되는 성질을 공유한다. 이들의 높은 친화력은 기존에 생각하던 가능한 길이보다 훨씬 짧은 올리고뉴클레오티드를 개발할 수 있도록 하였다. 2′-O-Me, 2′-MOE, 2′-F, 또는 LNA를 올리고뉴클레오티드에 도입하기 위한 합성법은 DNA 또는 RNA 합성과 양립가능하여, DNA 또는 RNA와의 키메라가 쉽게 얻어질 수 있다. 이러한 양립가능성은 화학적으로 변형된 올리고뉴클레오티드가 특정 용도에 미세 조정될 수 있도록 한다. Chemical modifications can also improve efficacy and selectivity by increasing the binding affinity of the nucleic acid to its complementary sequence. Widely used modifications are 2′-O-methyl (2′-O-Me), 2′-fluoro (2′-F), and 2′-O-methoxyethyl (2′-MOE) RNA. Additionally, higher affinity can be achieved by modifying the oligonucleotide with a locked nucleic acid (LNA) containing a methylene bridge between the 2' and 4' positions of the ribose. The bridge anchors the ribose ring in an ideal structure capable of achieving high affinity and binding to complementary sequences. Related bridged nucleic acid (BNA) compounds have been developed and share these desirable properties. Their high affinity made it possible to develop oligonucleotides much shorter than previously thought possible. Synthetic methods for introducing 2'-O-Me, 2'-MOE, 2'-F, or LNA into oligonucleotides are compatible with DNA or RNA synthesis, so that chimeras with DNA or RNA can be easily obtained. This compatibility allows chemically modified oligonucleotides to be fine-tuned for specific applications.
지난 10년 동안, 이중 가닥 siRNA는 유전자 발현을 억제하기 위한 수단으로 광범위하게 활용되었다. 이중 가닥 RNA가 세포에 들어가면, RISC의 단백질 기작에 결합한다. 유전자를 결손시키기 위해 사용되는 합성된 RNA는 일반적으로 19 내지 22 bp의 이중 가닥이다. 이 길이는 안정한 이중 가닥을 형성하여 RISC에 의해 인식되기에 충분하면서도 30 pb 이상의 길이의 이중 가닥이 유발하는 강한 인터페론 반응을 회피하기에 충분히 짧다. Over the past decade, double-stranded siRNA has been widely utilized as a means to suppress gene expression. When double-stranded RNA enters the cell, it binds to the protein machinery of RISC. The synthesized RNA used to delete genes is generally double stranded, 19 to 22 bp. This length is sufficient to form a stable duplex to be recognized by RISC, yet short enough to avoid the strong interferon response elicited by duplexes longer than 30 pb.
2001년에 포유동물 세포에서 유전자 결실에 관한 첫 번째 논문이 발표된 후, siRNA는 기능을 시험하기 위한 수천가지의 실험적 연구의 대상이 되었다. 안티센스 올리고뉴클레오티드가 유전자 결실에 계속 사용되는 동안, siRNA의 강력한 특성과 상대적으로 용이한 활성 siRNA의 확인법으로 인해 많은 실험실에서 선호하는 결실 수단이 되었다. Since the first paper on gene deletion in mammalian cells was published in 2001, siRNA has been the subject of thousands of experimental studies to test its function. While antisense oligonucleotides continue to be used for gene deletion, the powerful properties of siRNA and the relative ease of identifying active siRNA have made them the preferred method of deletion in many laboratories.
배양된 세포에서 변형되지 않은 이중 가닥 RNA는 놀랍게도 안정하고 화학적으로 변형된 siRNA는 일반적으로 유전자 발현의 결손에 필수적이지는 않다. 그러나 in vivo에서는 변형되지 않은 siRNA는 활성이 높지 않고, 화학적 변형을 통해 이들의 특성을 현저히 개선할 수 있다. 화학적으로 변형된 siRNA는 개선된 핵산 분해 효소 안정성 및 활성의 지속 시간의 증가를 보일 수 있다. 변형되지 않은 RNA는 또한 빠르게 제거되고, 화학적 변형, 담체와의 복합체 형성, 및 질환 표적에 국소적 전달은 개선된 in vivo에서의 결과를 달성하는 데에 도움을 줄 수 있다. In cultured cells, unmodified double-stranded RNA is surprisingly stable, and chemically modified siRNA is generally not essential for defects in gene expression. However, in vivo, unmodified siRNA does not have high activity, and its properties can be significantly improved through chemical modification. Chemically modified siRNAs can show improved nuclease stability and increased duration of activity. Unmodified RNA is also rapidly cleared, and chemical modification, complex formation with carriers, and localized delivery to disease targets can help achieve improved in vivo outcomes.
본원 발명에서 사용되는 올리고뉴클레오티드는 통상적으로 공지된 기술, 예를 들어, 고체상 합성을 통해 생산될 수 있다. 이러한 합성법에 사용되는 장비는 시중에서 구할 수 있으며, 합성을 위해 다른 수단들 역시 부가될 수 있다. Oligonucleotides used in the present invention can be produced through commonly known techniques, for example, solid phase synthesis. The equipment used in this synthesis method is commercially available, and other means may also be added for synthesis.
FOXM1 핵산에 코딩되는 단백질의 발현 또는 활성을 억제하기 위한 억제성 핵산의 세포로의 치료학적 투여는 당해 기술 분야에서 통상의 지식을 가진 자에게 알려진 임의의 핵산을 투여하는 방법을 포함한다. 암을 치료하기 위하여, 경구 투여 또는 주사, 또는 둘 모두를 통해 전달할 수 있다. 핵산 분자는 당해 기술 분야에서 공지된 다양한 방법을 통해 세포에 전달될 수 있으며, 예를 들어, 이온영동, 또는 하이드로겔, 사이클로덱스트린, 생분해성 나노캡슐, 및 생접합성 미소구체와 같은 다른 담체로의 병합, 또는 단백질성 벡터에 의해 리포좀으로의 캡슐화를 이용할 수 있다. Therapeutic administration of inhibitory nucleic acids to cells to inhibit the expression or activity of proteins encoded by FOXM1 nucleic acids includes administering any nucleic acid known to those skilled in the art. To treat cancer, it can be delivered via oral administration or injection, or both. Nucleic acid molecules can be delivered to cells through a variety of methods known in the art, such as iontophoresis or transfer to other carriers such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioconjugate microspheres. Incorporation, or encapsulation into liposomes by proteinaceous vectors can be used.
특정 구체예에서, FOXM1 핵산에 코딩되는 단백질의 발현 또는 활성을 억제하기 위한 억제성 핵산은 세포 기관, 세포, 조직, 종양, 또는 생물에 피하, 피내, 근육내, 정맥내, 복강내 주사 등으로 투여될 수 있다. In certain embodiments, the inhibitory nucleic acid to inhibit the expression or activity of the protein encoded by the FOXM1 nucleic acid is administered to a cell organelle, cell, tissue, tumor, or organism by subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal injection, etc. may be administered.
상기의 억제성 핵산 또는 다른 활성 성분이 투여에 적합한 약학 조성물에 첨가될 수 있다. 예를 들어, 약학 조성물은 FOXM1 핵산에 의해 코딩되는 단백질의 발현 또는 활성을 감소시킬 수 있는 하나 이상의 억제성 핵산 및 약학적으로 허용가능한 담체를 포함한다. The above inhibitory nucleic acids or other active ingredients may be added to pharmaceutical compositions suitable for administration. For example, the pharmaceutical composition includes one or more inhibitory nucleic acids capable of reducing the expression or activity of the protein encoded by the FOXM1 nucleic acid and a pharmaceutically acceptable carrier.
상기의 억제성 핵산은 서방형 조성물로 제공될 수 있다. 즉시 방출 또는 서방향 조성물은 치료될 환자의 조건에 따라 결정될 수 있다. 만약 환자의 상태가 급성 또는 과급성 질환인 경우, 치료는 즉시 방출형을 사용하여야 하고, 반면 예방적 또는 장기적 치료인 경우에는 서방형 조성물이 적합하다. The inhibitory nucleic acid may be provided as a sustained-release composition. Immediate or sustained release compositions may be determined depending on the condition of the patient being treated. If the patient's condition is an acute or hyperacute disease, the treatment should use an immediate-release composition, whereas in the case of preventive or long-term treatment, a sustained-release composition is suitable.
본 발명의 FoxM1 shRNA 또는 FoxM1 억제제 또는 점 돌연변이를 포함하는 FoxM1 단백질을 유효성분으로 포함하는 항암제는 특히, FoxM1의 발현이 과다한 다양한 암종, 폐암(Wang, I-Ching et al. PLoS One 4.8 (2009): e6609), 대장암(Yoshida, Yuichi et al., Gastroenterology 132.4 (2007) 1420-1431), 전립선암(Kalin, Tanya V. et al., Cancer research 66.3 (2006) 1712-1720), 유방암(Millour, Julie and E. W. Lam. Breast Cancer Research 12.1 (2010) 1-1) 및 뇌암(Liu, Mingguang et al., Cancer research 66.7 (2006) 3593-3602) 등의 각종 고형암 및 백혈병 등의 예방 및 치료에 이용될 수 있으며(Nakamura, Satoki et al., Carcinogenesis 31.11 (2010): 2012-2021), 또한 전이성 고형암의 예방 및 치료(Li, Lijun et al., Oncotarget 8.19 (2017): 32298)에도 이용될 수 있다.The anticancer agent containing FoxM1 shRNA or FoxM1 inhibitor or FoxM1 protein containing a point mutation of the present invention as an active ingredient, especially various carcinomas and lung cancer with excessive expression of FoxM1 (Wang, I-Ching et al. PLoS One 4.8 (2009) : e6609), colon cancer (Yoshida, Yuichi et al., Gastroenterology 132.4 (2007) 1420-1431), prostate cancer (Kalin, Tanya V. et al., Cancer research 66.3 (2006) 1712-1720), breast cancer (Millour , Julie and E. W. Lam. Breast Cancer Research 12.1 (2010) 1-1) and various solid cancers such as brain cancer (Liu, Mingguang et al., Cancer research 66.7 (2006) 3593-3602) and leukemia. It can be used (Nakamura, Satoki et al., Carcinogenesis 31.11 (2010): 2012-2021), and can also be used for the prevention and treatment of metastatic solid cancer (Li, Lijun et al., Oncotarget 8.19 (2017): 32298). .
본원에 참조된 문헌 및 연구에 대한 언급은 임의의 전술한 내용이 선행 기술 분야와 관련 있다는 인정으로서 의도되진 않는다. 이들 문헌의 내용에 대한 모든 언급은 출원인에게 이용가능한 정보를 기반으로 하며, 이들 문헌의 내용에 대한 정확성에 관한 어떠한 인정으로도 간주되지 않는다.Reference to literature and studies referenced herein is not intended as an admission that any foregoing content is relevant to prior art. All references to the content of these documents are based on information available to the applicant and are not considered as any admission as to the accuracy of the content of these documents.
후술한 내용은 당해 기술 분야의 당업자가 다양한 구현예들을 만들고 이용할 수 있게 하기 위해 제공된다. 구체적인 장치, 기법 및 이용에 관한 설명은 단지 예로서 제공된다. 본원에 기술된 예에 대한 다양한 변형들이 당해 기술 분야의 당업자들에게 용이하게 자명할 것이며, 본원에 정의된 일반적인 원리는 다양한 구현예들의 사상 및 범위로부터 이탈하지 않으면서 다른 예 및 활용에 적용될 수 있다. 따라서, 다양한 구현예들은 본원에 기술되고 보여진 예들로 한정하고자 하는 것은 아니며, 청구항과 일치하는 범위에 부합되는 것으로 의도된다.The following description is provided to enable those skilled in the art to make and use various implementations. Descriptions of specific devices, techniques and uses are provided by way of example only. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and uses without departing from the spirit and scope of the various embodiments. . Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but are intended to be accorded the scope consistent with the claims.
실시예Example
실시예 1Example 1
<실시예 1.1> 전이성 폐암세포에서 FoxM1과 활성형 PLK1의 임상적 연관성 및 TGF-β에 의해 유도된 암전이 조건에서 FoxM1과 활성형 PLK1의 발현 분석<Example 1.1> Clinical correlation between FoxM1 and active PLK1 in metastatic lung cancer cells and expression analysis of FoxM1 and active PLK1 in cancer metastasis conditions induced by TGF-β
최근 연구(Shin S.B. et al., Oncogene 39 (2020) 767-785)에서 비소세포폐암 세포에서 PLK1에 의해서 유도된 암전이 과정동안 ECM-adhesion 관련 유전자들의 변화를 관찰하였다(도 1A). 본 발명자는 비소세포폐암에서 FoxM1과 PLK1의 상관관계를 분석하고 임상적인 유의성을 검토하기 위하여 cBioPortal에서 등록된 빅데이터를 이용하여 폐선암 환자에서 FoxM1과 PLK1의 mRNA 발현간의 상관관계를 Spearman과 Pearson의 상관계수를 분석한 결과 유의적인 범위에서 두 인자의 발현에는 양의 상관 관계가 있음을 확인하였다(도 1A). 뿐만 아니라 다른 세포주기에 관련된 인자들도 함께 그 상관성을 분석한 결과, FoxM1과 MKI67의 발현이 PLK1발현과 높은 상관계수를 보이는 인자로 분석되었다(도 1B, 1C). In a recent study (Shin S.B. et al., Oncogene 39 (2020) 767-785), changes in ECM-adhesion-related genes were observed during the cancer metastasis process induced by PLK1 in non-small cell lung cancer cells (Figure 1A). In order to analyze the correlation between FoxM1 and PLK1 in non-small cell lung cancer and examine its clinical significance, the present inventor used big data registered in cBioPortal to determine the correlation between the mRNA expression of FoxM1 and PLK1 in lung adenocarcinoma patients using Spearman and Pearson's method. As a result of analyzing the correlation coefficient, it was confirmed that there was a positive correlation in the expression of the two factors in a significant range (Figure 1A). In addition, as a result of analyzing the correlation with other cell cycle-related factors, the expression of FoxM1 and MKI67 was analyzed as a factor showing a high correlation coefficient with PLK1 expression (Figures 1B, 1C).
다음으로는 비소세포폐암 환자에서 FOXM1과 PLK1이 임상적 연관성을 확인하기 위해서 FOXM1과 PLK1의 발현량에 따른 환자의 전체 생존율을 빅데이터를 분석을 통해서 확인하였다(도 1D, E, F). 비소세포폐암 환자에서 PLK1과 FOXM1 발현이 높은 환자의 생존율은 PLK1과 FOXM1의 발현이 낮은 환자의 생존율에 비해 현저히 낮은 것을 확인할 수 있었다(도 1D). 비소세포폐암은 폐선암(adenocarcinoma)과 편평세포폐암 (Squamous lung cell carcinoma) 나누는 데, 이를 나누어서 분석한 결과에 따르면, 폐선암 환자에서, PLK1과 FOXM1 발현이 높은 환자의 생존율은 PLK1과 FOXM1의 발현이 낮은 환자의 생존율에 비해 현저히 낮은 것을 확인할 수 있었다(도 1E). 특히 폐선암환자 중 전이암이 관찰되는 stage 3-4인 환자 대상으로 재분석한 결과, PLK1과 FOXM1 발현이 높은 환자의 생존율은 PLK1과 FOXM1의 발현이 낮은 환자의 생존율에 비해 현저히 낮은 것을 확인할 수 있었다(도 1F). 추가적으로 폐선암 환자를 stage 별로 FoxM1과 PLK1의 발현정도를 heatmap으로 분석한 결과, 정상 조직에 비해 암환자에서, 그리고 stage 1보다는 stage 2-4의 조직에서, PLK1과 FOXM1 발현이 높음을 확인하였다(도 1G). Next, in order to confirm the clinical correlation between FOXM1 and PLK1 in non-small cell lung cancer patients, the overall survival rate of patients according to the expression level of FOXM1 and PLK1 was confirmed through big data analysis (Figure 1D, E, F). In non-small cell lung cancer patients, the survival rate of patients with high expression of PLK1 and FOXM1 was confirmed to be significantly lower than that of patients with low expression of PLK1 and FOXM1 (Figure 1D). Non-small cell lung cancer is divided into adenocarcinoma and squamous lung cell carcinoma. According to the results of analysis of these divisions, in lung adenocarcinoma patients, the survival rate of patients with high expression of PLK1 and FOXM1 was higher than that of patients with high expression of PLK1 and FOXM1. It was confirmed that the survival rate was significantly lower than the low patient survival rate (Figure 1E). In particular, as a result of re-analysis of patients with stage 3-4 lung adenocarcinoma where metastatic cancer was observed, it was confirmed that the survival rate of patients with high PLK1 and FOXM1 expression was significantly lower than the survival rate of patients with low expression of PLK1 and FOXM1. (Figure 1F). Additionally, as a result of analyzing the expression levels of FoxM1 and PLK1 in lung adenocarcinoma patients by stage using a heatmap, it was confirmed that PLK1 and FOXM1 expression was higher in cancer patients compared to normal tissues and in stage 2-4 tissues rather than stage 1 ( Figure 1G).
<실시예 1.2> TGF-β에 의해 유도한 전이성 폐암세포에서 활성형 PLK1에 의한 FoxM1의 인산화 및 신규 인산화 부위 확인<Example 1.2> Phosphorylation of FoxM1 by activated PLK1 and identification of new phosphorylation sites in metastatic lung cancer cells induced by TGF-β
비소세포폐암 환자를 대상으로 FoxM1과 PLK1의 발현과 생존율, 암 단계에 따른 발현분석에서 FoxM1과 PLK1의 발현이 전이암에서 높아 생존율을 낮추는 원인으로 작용할 가능성이 임상데이터에서 관찰된 바, 본 발명자들은 전이암 모델에서 FoxM1과 PLK1의 기능을 관찰하고자 하였다. In the analysis of the expression and survival rate of FoxM1 and PLK1 in patients with non-small cell lung cancer, and expression according to cancer stage, the present inventors observed in clinical data that the expression of FoxM1 and PLK1 is high in metastatic cancer, which may lower the survival rate. We sought to observe the functions of FoxM1 and PLK1 in a metastatic cancer model.
이를 위하여, TGF-β를 처리하여 암전이 세포모델인 상피간엽이행(EMT)을 유도하는 과정에서 PLK1과 FoxM1의 발현 변화와 PLK1의 활성화여부를 관찰하고자 비소세포폐암 세포주 A549, NCI-H358, NCI-H460 세포주에 TGF-β를 처리하여 상피간엽이행을 유도한 후 mRNA 발현량과 단백질 발현량을 분석하였다. To this end, to observe changes in the expression of PLK1 and FoxM1 and activation of PLK1 in the process of inducing epithelial-mesenchymal transition (EMT), a cancer metastasis cell model, by treating TGF-β, non-small cell lung cancer cell lines A549, NCI-H358, and NCI -H460 cell line was treated with TGF-β to induce epithelial-mesenchymal transition, and the mRNA and protein expression levels were analyzed.
비소세포폐암 세포주 A549, NCI-H358, NCI-H460 세포주에 TGF-β를 5 ng/μl 처리한 군에서 간엽형질 마커인 CDH2, SNAl1 SNAI2의 mRNA 발현량 증가와 상피형질 마커인 CDH1의 mRNA 발현량 감소를 관찰하였다. 이 조건에서 대조군에 비하여 PLK1과 FOXM1의 mRNA 발현량의 증가를 관찰할 수 있었다(도 2A, 2B, 2C). 또한 vimentin, PLK1, E-cadherin과 N-cadherin의 단백질량도 mRNA 발현량과 같은 결과의 양상을 보여주었고 특히, PLK1의 활성형인 T210 잔기에서의 인산화 단백질 양이 대조군에 비해 TGF-β 처리군에서 높게 증가되는 것을 관찰할 수 있었다(도 2D). 단백질 양의 상대적 변화를 그래프로 표시하였다 (도2D, 오른쪽 그림).In the non-small cell lung cancer cell lines A549, NCI-H358, and NCI-H460 cell lines treated with 5 ng/μl TGF-β, the mRNA expression level of CDH2, SNA11, and SNAI2, mesenchymal markers, and the mRNA expression level of CDH1, an epithelial marker, were increased. A decrease was observed. Under these conditions, an increase in the mRNA expression levels of PLK1 and FOXM1 was observed compared to the control group (Figures 2A, 2B, 2C). In addition, the protein amounts of vimentin, PLK1, E-cadherin, and N-cadherin showed the same results as the mRNA expression levels. In particular, the amount of phosphorylated protein at the T210 residue, the active form of PLK1, was higher in the TGF-β treatment group compared to the control group. An increase could be observed (Figure 2D). The relative change in protein amount was displayed graphically (Figure 2D, right picture).
PLK1과 FoxM1과의 상관성 분석을 위하여, FoxM1의 인산화가 EMT에 의존하는지 여부를 조사하기 위해 A549 및 NCI-H460 세포에 TGF-β를 처리한 세포에 탈인산화효소(Phosphatase, CIP)를 처리하여 p-FoxM1, p-PLK1, p-TCTP의 인산화 정도를 겔 지연과 인산화-항체를 활용하여 면역침강법과 면역블랏법을 이용하여 분석하였다. 연구결과, 탈인산화효소 처리로 TGF-β 처리에 의해 상향 이동된 PLK1, TCTP 및 FoxM1의 밴드의 이동성을 지연시킴을 알 수 있었다. 또한, 인산화 항체를 활용하여 검토한 결과, p-FoxM1 Ser, p-PLK1 T210 및 p-TCTP S46의 수준이 탈인산화효소 처리에 의해 감소됨을 확인하였다(도 2E, 2F). 이는 TGF-β 처리로 유도된 EMT동안 FOXM1과 PLK1이 인산화됨을 알 수 있었다. 따라서, FoxM1과 PLK1은 비소세포폐암 전이가 유도되는 환경에서 그 발현이 높으며 PLK1 활성 증가 및 FoxM1의 인산화와 비례 관계가 있어 암전이에서 이들인자의 인산화와의 상관관계가 있음을 알 수 있었다.To analyze the correlation between PLK1 and FoxM1, and to investigate whether phosphorylation of FoxM1 is dependent on EMT, A549 and NCI-H460 cells treated with TGF-β were treated with phosphatase (CIP) to determine p The degree of phosphorylation of -FoxM1, p-PLK1, and p-TCTP was analyzed using immunoprecipitation and immunoblotting using gel retardation and phosphorylation-antibodies. As a result of the study, it was found that dephosphorylation enzyme treatment delayed the mobility of the bands of PLK1, TCTP, and FoxM1, which were upwardly moved by TGF-β treatment. In addition, as a result of examining using a phosphorylation antibody, it was confirmed that the levels of p-FoxM1 Ser, p-PLK1 T210, and p-TCTP S46 were reduced by dephosphorylation enzyme treatment (Figures 2E, 2F). This showed that FOXM1 and PLK1 were phosphorylated during EMT induced by TGF-β treatment. Therefore, the expression of FoxM1 and PLK1 is high in an environment where non-small cell lung cancer metastasis is induced, and there is a proportional relationship with increased PLK1 activity and phosphorylation of FoxM1, indicating a correlation with phosphorylation of these factors in cancer metastasis.
<실시예 1.3> FoxM1의 인산화 및 비인산화 점 돌연변이형 발현을 위한 렌티바이러스 시스템 구축 <Example 1.3> Construction of a lentiviral system for expression of phosphorylated and non-phosphorylated point mutant forms of FoxM1
본 발명자들은 FoxM1의 인산화 및 비인산화 유전자 발현을 위한 lentivirus system 구축을 위하여 pLVX-TRE3G-eRFP 에, NotI과 EcoRI으로 cutting후, FoxM1 원형(WT) 플라스미드를 5' -ACGGGGCCCATGAAAACTAGCCCCCGTCG-3'(forward primer)와 5'-ACGGGAATTCCTACTGTAGCTCAGGAATAA-3'(reverse primer)의 프라이머를 이용하여 PCR로 증폭시킨 후 이를 NotI과 EcoRI으로 자른 후, 벡터 pLVX-TRE3G-eRFP에 서브클로닝하였다. 렌티 바이러스 시스템을 구축하기 위해서 pCMV-VSV.G와 pCMV-Δ8.2, pLVX-TRE3G-eRFP-Target 또는 pLVX-Tet3G DNA를 HEK293 세포에서 발현시키고자 형질감염하여 렌티 바이러스를 발현시켜 바이러스를 모은 후 이를 암세포에 사용하고자 하였다. 형질감염 후 바이러스 배양액은 12시간 간격으로 72시간까지 모았으며, 0.2 mm 필터로 배양액을 필터하고 18000 rpm, 4℃, 90분 동안 원심분리하였다. 상등액은 버리고 TNE 완충액으로 모아진 바이러스를 모은 후 4℃에 보관하여 다음날부터 암세포 감염에 사용하였다. In order to construct a lentivirus system for expression of phosphorylated and non-phosphorylated FoxM1 genes, the present inventors cut pLVX-TRE3G-eRFP with NotI and EcoRI, and then added the original FoxM1 (WT) plasmid to 5' -ACGGGGCCCATGAAAACTAGCCCCCGTCG-3' (forward primer). It was amplified by PCR using the primers 5'-ACGGGAATTCCTACTGTAGCTCAGGAATAA-3' (reverse primer), cut with NotI and EcoRI, and subcloned into vector pLVX-TRE3G-eRFP. To construct a lentivirus system, pCMV-VSV.G, pCMV-Δ8.2, pLVX-TRE3G-eRFP-Target or pLVX-Tet3G DNA was transfected to express lentivirus in HEK293 cells, and then viruses were collected. The intention was to use this on cancer cells. After transfection, the virus culture was collected at 12-hour intervals for up to 72 hours, filtered with a 0.2 mm filter, and centrifuged at 18000 rpm at 4°C for 90 minutes. The supernatant was discarded, and the viruses collected in TNE buffer were stored at 4°C and used to infect cancer cells from the next day.
<실시예 1.4> 렌티바이러스를 이용한 FoxM1의 활성형 및 비활성형 유전자 발현 세포 선별 후 상피간엽이행 효과 평가<Example 1.4> Evaluation of the effect of epithelial-mesenchymal transition after selection of cells expressing active and inactive genes of FoxM1 using lentivirus
점 돌연변이를 포함하는 FoxM1 단백질의 암세포 상피간엽이행 조절효과를 검토하기 위하여, 폐암세포에 FoxM1의 인산화 및 비인산화 점 돌연변이체가 발현시키는 렌티바이러스를 감염시키고자 다음과 같이 암세포를 배양하였다. To examine the regulatory effect of FoxM1 protein containing point mutations on cancer cell epithelial-mesenchymal transition, lung cancer cells were cultured as follows to infect lung cancer cells with lentivirus expressing phosphorylated and non-phosphorylated point mutants of FoxM1.
폐암 세포인 A549에 FoxM1의 원형 및 인산화와 비인산화된 점 돌연변이체를 발현시키는 안정화된 세포주를 구축하기 위해서 먼저 pLVX-Tet3G 발현성 렌티바이러스를 감염시키고 G418을 5일 동안 처리하여 감염된 세포를 선별하였다. 이렇게 선별된 A549Tet3G 세포에 FoxM1의 원형 및 인산화된 점 돌연변이형(S25E, S361E, S715E)와 비인산화된 점 돌연변이형(S25A, S361A, S715A)을 발현시키는 렌티바이러스를 감염시킨 후 퓨로마이신을 48시간동안 처리하여 안정화된 세포주를 구축하였다. 구축된 세포에 2μg/ml으로 독시사이클린을 처리하여 FoxM1의 원형 및 인산화와 비인산화형의 점 돌연변이형을 발현을 유도 후 각각의 FoxM1의 점 돌연변이체가 잘 발현되었는지 mRNA 발현량과 단백질 발현량을 확인하였다. 도 4A, 도 4B에서와 같이, FoxM1의 원형 및 각각의 점 돌연변이형이 잘 발현되고 있는 것을 mRNA 발현량과 단백질 발현량을 통해서 확인하였다. 인산화 부위 S25에서, 인산화형의 FoxM1이 발현되는 폐암세포에서 비인산화형 FoxM1를 발현하고 있는 세포에서 보다 N-cadherin, Vimentin의 발현이 높고 E-cadherin의 발현이 낮은 것을 관찰하였다. 또한 이러한 FoxM1의 인산화 및 비인산화 점 돌연변이체 발현이 세포의 증식에 영향을 주는지 확인해 본 결과, 세포의 증식에는 큰 영향을 미치지 않는 것으로 관찰되었다(도 4C).To construct a stable cell line expressing the original form and phosphorylated and non-phosphorylated point mutants of FoxM1 in lung cancer cell A549, the infected cells were first infected with pLVX-Tet3G-expressing lentivirus and treated with G418 for 5 days to select infected cells. . The selected A549Tet3G cells were infected with lentivirus expressing the original and phosphorylated point mutant forms (S25E, S361E, S715E) and non-phosphorylated point mutant forms (S25A, S361A, S715A) of FoxM1, and then treated with puromycin for 48 hours. A stabilized cell line was constructed by treatment for a period of time. The constructed cells were treated with doxycycline at 2 μg/ml to induce expression of the original form and phosphorylated and non-phosphorylated point mutant forms of FoxM1, and then the mRNA and protein expression levels were checked to see whether each point mutant of FoxM1 was well expressed. As shown in Figures 4A and 4B, it was confirmed through the mRNA and protein expression levels that the original form and each point mutant form of FoxM1 were well expressed. At the phosphorylation site S25, we observed higher expression of N-cadherin and Vimentin and lower expression of E-cadherin in lung cancer cells expressing phosphorylated FoxM1 than in cells expressing non-phosphorylated FoxM1. In addition, as a result of checking whether the expression of these phosphorylated and non-phosphorylated point mutants of FoxM1 affected cell proliferation, it was observed that there was no significant effect on cell proliferation (Figure 4C).
이상의 결과로, FoxM1 S25 잔기의 인산화 점 돌연변이체(S25E)는 상피간엽이행을 촉진하는 효과를 시사하였다.The above results suggested that the phosphorylated point mutant (S25E) of the FoxM1 S25 residue had an effect in promoting epithelial-mesenchymal transition.
<실시예 1.5> FoxM1의 인산화 및 비인산화 점 돌연변이를 포함하는 단백질에 의한 전이성 촉진 및 억제 효과 평가<Example 1.5> Evaluation of metastatic promotion and inhibition effects by proteins containing phosphorylation and non-phosphorylation point mutations of FoxM1
FoxM1 원형, 인산화 및 비인산화 점 돌연변이체 단백질을 발현시키는 폐암세포 A549에서 이들 점 돌연변이체가 암세포의 이동성에 미치는 효과를 관찰하기 위해 세포이동성 실험(migration assay)을 실시하였다. A migration assay was performed to observe the effect of these point mutants on the mobility of cancer cells in lung cancer cells A549 expressing the original, phosphorylated, and non-phosphorylated FoxM1 point mutant proteins.
구체적으로, 각각의 FoxM1 원형, 인산화 및 비인산화 점 돌연변이체 단백질을 발현시키는 폐암세포 A549를 5 x 104 개의 세포를 8.0 μm, 24 well insert에 분주하고 24 well plate에 10% 혈청(FBS)이 포함된 배지를 분주한 뒤 inset를 넣어 주었다. 양성 대조군의 경우, 5 ng/ml TGF-β가 포함된 RPMI 1640(10% FBS)를 0.5 ml 분주하여 사용하였다. 세포분주 72시간 후, 4% 파라포름알데히드(paraformaldehyde) 500 ㎕를 분주하고 1XPBS로 3회 세척하여 0.05% 크리스탈-바이올렛 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도(intensity)를 Odyssey infrared imaging system을 이용하여 측정하였다. 대조군의 염색된 정도(intensity)를 1이라고 할 때 각 실험군에서의 상대적 염색된 정도를 산출하여 그래프를 표시하였다.Specifically, 5 x 10 cells of lung cancer cells A549 expressing each original FoxM1, phosphorylated and non-phosphorylated point mutant protein were dispensed into 8.0 μm, 24 well inserts, and 10% serum (FBS) was added to the 24 well plate. The resulting medium was dispensed and then added to the inset. For the positive control, 0.5 ml of RPMI 1640 (10% FBS) containing 5 ng/ml TGF-β was used. 72 hours after cell distribution, 500 ㎕ of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the intensity of staining was measured using the Odyssey infrared imaging system. Assuming that the intensity of staining in the control group was 1, the relative staining intensity in each experimental group was calculated and displayed on a graph.
연구 결과, FoxM1 인산화 점 돌연변이체 단백질을 발현시킨 실험군에서 양성대조군인 TGF-β 처리군과 유사하거나 더 높게 염색강도(intensity) 수치가 대조군 대비 6배까지 증가하였으며, 상대적으로 FoxM1 인산화 돌연변이체에 비해 비인산화 점 돌연변이체 실험군은 염색강도가 낮은 것을 관찰할 수 있었다(도 4D). 하지만 FoxM1의 S361번, S715번 인산화 돌연변이체는 암세포 이동성에 영향을 주지 않는 것을 관찰할 수 있었다. 이상의 결과로 FoxM1 S25잔기의 인산화 점 돌연변이체는 폐암세포의 이동성을 촉진하며, 반면에 FoxM1 S25잔기의 비인산화 점 돌연변이체는 폐암세포의 이동성을 억제하는 효과가 뛰어남을 알 수 있었다.As a result of the study, in the experimental group expressing the FoxM1 phosphorylated point mutant protein, the staining intensity increased up to 6 times compared to the control group, similar to or higher than the positive control group, the TGF-β treatment group, and relatively compared to the FoxM1 phosphorylated mutant protein. The non-phosphorylated point mutant experimental group was observed to have low staining intensity (Figure 4D). However, it was observed that the S361 and S715 phosphorylation mutants of FoxM1 did not affect cancer cell mobility. The above results showed that the phosphorylated point mutant of the FoxM1 S25 residue promoted the mobility of lung cancer cells, while the non-phosphorylated point mutant of the FoxM1 S25 residue had an excellent effect in suppressing the mobility of lung cancer cells.
<실시예 1.6> FoxM1의 인산화부위 및 비인산화 점 돌연변이를 포함하는 단백질에 의한 침습성 촉진 및 억제 효과 평가<Example 1.6> Evaluation of invasiveness promotion and inhibition effects by proteins containing phosphorylation sites and non-phosphorylation point mutations in FoxM1
FoxM1 원형, 인산화 및 비인산화 점 돌연변이체 단백질을 발현시키는 폐암세포 A549에서 이들 점 돌연변이체가 암세포의 침습성에 미치는 효과를 관찰하기 위해 마트리겔을 이용한 침습성 실험(invasion assay)을 실시하였다. An invasion assay using Matrigel was performed to observe the effect of these point mutants on the invasiveness of cancer cells in lung cancer cells A549 expressing the original, phosphorylated and non-phosphorylated FoxM1 point mutant proteins.
구체적으로, 4℃에서 16~20시간동안 마트리겔을 완전히 녹인 후 마트리겔을 1 mg/ml이 되도록 차가운 serum free RPMI 1640(4℃)으로 희석하였다. 이를 8.0 mm 24 well 인서트(insert)에 100 μl의 마트리겔 혼합물(1 mg/ml)를 넣고 37℃ 배양기에서 12-20시간 동안 굳혀주었다. 굳은 마트리겔 인서트에 각각의 FoxM1 원형, 인산화 및 비인산화 점 돌연변이체 단백질을 발현시키는 폐암세포 A549들을 1X105 cells/well의 세포수로 serum free RPMI 1640 (36℃)에 희석하여 인서트에 분주하였다. 여기에 36℃의 따뜻한 RPMI 1640 (10% FBS)을 0.5 ml/well씩 분주하였다. 양성 대조군의 경우 5 ng/ml TGF-β가 포함된 36℃ RPMI 1640(10% FBS)를 0.5 ml 분주하여 사용하였다. 이 후 3일에 한 번씩 배지를 교환해주고 침습되는 정도를 관찰하였으며, 암세포의 침습이 충분히 일어난 것으로 관찰된 7일 차에 배지를 제거하고 1XPBS로 세척한 후, insert 안쪽의 cell들을 면봉으로 긁어 내었고, 1XPBS로 세척하여 인서트 내부에 세포와 마트리겔의 잔여물이 남지 않도록 제거하였다. 인서트 바깥 면이 있는 24 well에 4% 파라포름알데하이드 500μl를 분주하고 5분간 실온에서 배양한 후 1XPBS로 3회 세척하여, 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도를 DMSO로 녹인 후 590nm에서 파장을 측정하였다. 대조군의 흡광도를 1이라고 할 때 각 실험군에서의 상대적 흡광도를 산출하여 그래프를 표시하였다.Specifically, Matrigel was completely dissolved at 4°C for 16 to 20 hours, and then Matrigel was diluted to 1 mg/ml with cold serum-free RPMI 1640 (4°C). 100 μl of Matrigel mixture (1 mg/ml) was added to an 8.0 mm 24 well insert and hardened in a 37°C incubator for 12-20 hours. Lung cancer cells A549 expressing each FoxM1 original, phosphorylated and non-phosphorylated point mutant protein were diluted in serum free RPMI 1640 (36°C) at a cell number of 1X10 5 cells/well and dispensed onto the hardened Matrigel insert. Here, 0.5 ml/well of warm RPMI 1640 (10% FBS) at 36°C was dispensed. For the positive control, 0.5 ml of 36°C RPMI 1640 (10% FBS) containing 5 ng/ml TGF-β was used. After that, the medium was changed once every three days and the degree of invasion was observed. On the 7th day, when it was observed that cancer cell invasion had sufficiently occurred, the medium was removed, washed with 1XPBS, and the cells inside the insert were scraped off with a cotton swab. and washed with 1XPBS to remove any remaining cells and matrigel inside the insert. 500 μl of 4% paraformaldehyde was dispensed into 24 wells on the outer side of the insert, incubated at room temperature for 5 minutes, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the degree of staining was dissolved in DMSO and the wavelength was measured at 590 nm. Assuming that the absorbance of the control group was 1, the relative absorbance of each experimental group was calculated and displayed on a graph.
연구 결과, FoxM1 인산화 점 돌연변이체 단백질을 발현시킨 실험군에서 양성대조군인 TGF-β 처리군과 유사하거나 더 높게 흡광도 수치가 대조군 대비 근 40배까지 증가하였으며 상대적으로 FoxM1 비인산화 점 돌연변이체 실험군은 상대적 흡광도가 더 낮아진 것을 관찰할 수 있었다(도 4E). 따라서 FoxM1 S25잔기의 비인산화 점 돌연변이체는 폐암세포의 이동성과 침습성을 억제하는 효과가 뛰어남을 알 수 있었다.As a result of the study, the absorbance value in the experimental group expressing the FoxM1 phosphorylated point mutant protein was similar to or higher than that of the positive control group, the TGF-β treatment group, and increased to nearly 40 times that of the control group. It was observed that was further lowered (Figure 4E). Therefore, it was found that the non-phosphorylated point mutant of the FoxM1 S25 residue was highly effective in suppressing the mobility and invasiveness of lung cancer cells.
<실시예 1.7> FoxM1의 인산화 점 돌연변이와 3개 점 동시 돌연변이를 포함하는 단백질에 의한 이동성 효율의 효과 평가<Example 1.7> Evaluation of the effect of mobility efficiency by proteins containing phosphorylation point mutations and three point simultaneous mutations of FoxM1
FoxM1 원형(WT), 인산화 점 돌연변이체 각각 S25E, S361E, S715E 그리고 인산화 3개 점 동시 돌연변이체 EEE 단백질을 발현시키는 폐암세포 A549에서 이들 점 돌연변이체가 암세포의 이동성에 미치는 효과를 관찰하기 위해 상처 치유 분석 실험(Wound healing assay)을 실시하였다. Wound healing assay in lung cancer cells A549 expressing FoxM1 original (WT), phosphorylated point mutants S25E, S361E, and S715E, respectively, and three phosphorylated point mutant EEE proteins, respectively, to observe the effect of these point mutants on cancer cell mobility. An experiment (Wound healing assay) was performed.
구체적으로, 랜티바이러스 시스템에 의해 FoxM1 점 돌연변이체의 유전자를 발현하는 세포를 각각 1X105 cells/well로 6well plate에 분주한다. 분주 24시간 후, 200μl 파이펫 팁(pipette tip)을 사용하여 일정한 간격으로 스크래치를 낸다. 또한 음성 대조군(Mock)과 양성 대조군 5ng/ml의 TGF-β를 처리한 A549세포를 사용한다. 0h, 24h, 48h, 72h 간격으로 관찰하고 치유(Healing)되는 거리를 측정한다(도 4F). 또한 72h에서 대조군을 0%으로 전환하여 상대적 거리를 막대 그래프로 나타냈다(도 4G). Specifically, cells expressing the FoxM1 point mutant gene by the lantivirus system are distributed in a 6-well plate at 1X10 5 cells/well. 24 hours after dispensing, scratches are made at regular intervals using a 200μl pipette tip. Additionally, A549 cells treated with 5 ng/ml of TGF-β were used as negative control (Mock) and positive control. Observe at intervals of 0h, 24h, 48h, and 72h and measure the healing distance (Figure 4F). Additionally, the control group was converted to 0% at 72 h, and the relative distance was displayed as a bar graph (Figure 4G).
연구결과 FoxM1의 S25 잔기의 인산화 점 돌연변이체(S25E)가 발현되는 세포군에서는 대조군 대비 이동성이 현저히 증가된 것을 관찰할 수 있었고 72h에서 대조군을 0%로 전환하였을 때 S25E가 43% 임을 관찰할 수 있었다. 이는 양성 대조군인 TGF-β 처리 세포군(5ng/ml) 51%로 같은 증가 양상을 보여주었다. 인산화 점 돌연변이체(S361E)와 인산화 점 돌연변이체(S717E) 단백질을 발현하는 암세포의 이동성은 대조군과 대비 72h에서 각각 0.7%, 1.6%로 큰 차이가 없음을 관찰하였다. 또한, 3개 점 동시 돌연변이체(EEE)는 72h에서 15%로 점 돌연변이체(S25E)에 비해 이동성이 약하지만 대조군 대비 증가한 것을 관찰할 수 있었다. 따라서 FoxM1 S25 잔기의 인산화 점 돌연변이체(S25E)가 발현되는 세포군에서 암세포의 이동성이 증가되며 다른 두 개 인산화 점 돌연변이체 S361E와 S715E 단백질 발현되는 세포에서는 이동성이 크게 증가하지 않음을 알 수 있었다. 이상의 결과로 FoxM1 S25잔기의 인산화 점 돌연변이체는 폐암세포의 이동성을 촉진하며 효과가 뛰어남을 알 수 있었다.As a result of the study, in the cell group expressing the phosphorylated point mutant (S25E) of the S25 residue of FoxM1, significantly increased mobility was observed compared to the control group, and when the control group was changed to 0% at 72h, S25E was observed to be 43%. . This showed the same increase as the positive control TGF-β treated cell group (5ng/ml) of 51%. It was observed that the mobility of cancer cells expressing the phosphorylated point mutant (S361E) and phosphorylated point mutant (S717E) proteins was 0.7% and 1.6%, respectively, at 72h compared to the control group, showing no significant difference. In addition, the three point simultaneous mutant (EEE) was observed to have weaker mobility compared to the point mutant (S25E) at 15% at 72h, but increased compared to the control group. Therefore, it was found that the mobility of cancer cells was increased in the cell group expressing the phosphorylated point mutant (S25E) of the FoxM1 S25 residue, while the mobility was not significantly increased in the cells expressing the other two phosphorylated point mutant S361E and S715E proteins. The above results showed that the phosphorylated point mutant of the S25 residue of FoxM1 promotes the mobility of lung cancer cells and is highly effective.
<실시예 1.8> 동물모델에서 FoxM1의 인산화부위 및 비인산화 점 돌연변이를 포함하는 단백질에 의한 암세포의 전이성 및 종양형성능 평가<Example 1.8> Evaluation of metastatic and tumorigenic ability of cancer cells by protein containing phosphorylation site and non-phosphorylation point mutation of FoxM1 in animal model
본 발명자들은 동물모델에서 암세포의 종양 형성능을 관찰하기 위해 FoxM1 S25잔기의 인산화 및 비인산화 점 유전자가 발현되는 암세포를 이용하여 암세포의 종양형성 촉진 및 억제 효과를 평가하고자 하였다.In order to observe the tumorigenic ability of cancer cells in an animal model, the present inventors attempted to evaluate the effect of promoting and suppressing tumorigenesis of cancer cells using cancer cells expressing phosphorylated and non-phosphorylated genes of the FoxM1 S25 residue.
구체적으로, 점 돌연변이를 포함하는 FoxM1 단백질이 안정적으로 발현되는 A549 폐암세포(2X106 세포수)를 PBS에 넣어 마우스의 꼬리 정맥에 주사하여, 8주 동안 사육하고, 개복하여 장기에 암세포가 전이 및 종양형성능을 관찰하였다. 비교를 위해 대조군(Mock; 목적유전자가 발현되지 않는 렌티바이러스 처리군), FoxM1 단백질 발현되는 A549 폐암세포주 투여군(WT), 인산화 점 돌연변이체 FoxM1 단백질 발현되는 A549 폐암세포주 투여군(S25E), 비인산화 점 돌연변이체 FoxM1 단백질 발현되는 A549 폐암세포주 투여군(S25A)에 대해서 암세포 전이 및 종양형성 여부를 관찰하였다. 각 실험군 마다 5마리의 마우스에 대하여 실험을 실시하고, 폐에서의 전이성 암인 종양형성 빈도수를 측정하여 그래프로 표시하였다(도 5).Specifically, A549 lung cancer cells ( 2 Tumor formation ability was observed. For comparison, control group (Mock; lentivirus treatment group in which target gene is not expressed), group administered A549 lung cancer cell line expressing FoxM1 protein (WT), group administered A549 lung cancer cell line expressing phosphorylated point mutant FoxM1 protein (S25E), non-phosphorylated point group. Cancer cell metastasis and tumor formation were observed in the A549 lung cancer cell line administration group (S25A) expressing mutant FoxM1 protein. The experiment was performed on 5 mice for each experimental group, and the frequency of tumor formation, which is metastatic cancer in the lung, was measured and displayed in a graph (FIG. 5).
도 5에 나타낸 바와 같이, 대조군(Mock)과 FoxM1 원형 대비 인산화 점 돌연변이를 포함하는 FoxM1 단백질이 발현되는 A549 세포를 투여한 실험군에서는 종양형성능이 비교적 높은 것을 관찰하였다. FoxM1 인산화 점 돌연변이체 중 S325E 실험군에서는 생성된 종양의 수를 관찰했을 때 가장 높은 종양형성능을 확인할 수 있었다. 반대로 비인산화 점 돌연변이를 포함하는 FoxM1 단백질이 발현되는 A549 세포를 투여한 실험군에서는 종양형성능이 비교적 낮은 것을 관찰하였다(도 5A). H&E(Haematoxylin and eosin)과 Ki67 염색을 통해서 FoxM1 인산화 점 돌연변이체 실험군에서 암세포 증식정도가 높은 것을 알 수 있었다(도 5B, 5C). 이는 동물모델에서 FoxM1의 인산화 점 돌연변이체를 발현하는 암세포는 암전이성과 종양형성능을 촉진시키며, 반대로 비인산화 점 돌연변이체는 암전이성과 종양형성능을 억제하는 것을 확인할 수 있었다.As shown in Figure 5, it was observed that the tumorigenic ability was relatively high in the experimental group administered A549 cells expressing FoxM1 protein containing a phosphorylation point mutation compared to the control group (Mock) and the original FoxM1. Among the FoxM1 phosphorylation point mutants, the S325E experimental group showed the highest tumorigenicity when observed in the number of tumors generated. Conversely, in the experimental group administered A549 cells expressing FoxM1 protein containing a non-phosphorylation point mutation, a relatively low tumorigenic ability was observed (Figure 5A). Through H&E (Haematoxylin and eosin) and Ki67 staining, it was found that the degree of cancer cell proliferation was high in the FoxM1 phosphorylation point mutant experimental group (Figures 5B, 5C). In animal models, it was confirmed that cancer cells expressing phosphorylated point mutants of FoxM1 promoted cancer metastasis and tumorigenicity, while non-phosphorylated point mutants suppressed cancer metastasis and tumorigenic ability.
다음으로 폐 조직을 용해하여 상피간엽이행 마커의 단백질 수준을 관찰한 결과, FoxM1 인산화 점 돌연변이체의 실험군에서 중간엽 마커인 N-cadherin의 단백질 수준이 증가하였으며, E-cadherin의 수준은 감소하였다. 또한 면역회피에 관여하여 암세포의 종양형성능을 증가시킨다고 알려진 PD-L1의 발현도 각 실험군에서 확인한 결과, FoxM1 인산화 점 돌연변이체 실험군에서 발현이 높게 관찰되었다(도 5D). 추가로 단백질 수준뿐만 아니라 mRNA 수준에서도 상위와 같은 결과를 확인하였다(도 5E). 따라서 FoxM1의 인산화가 상피간엽이행과 전이성과 종양형성능을 증진시킴을 시사한다. 반면, 본 발명의 비인산화 점 돌연변이체 특히, S25A 실험군은 원형의 FoxM1 실험군보다 낮은 전이성과 암세포의 종양형성능을 억제하는 효과가 뛰어남을 알 수 있다. Next, the lung tissue was lysed and the protein level of the epithelial-mesenchymal transition marker was observed. As a result, the protein level of N-cadherin, a mesenchymal marker, increased, and the level of E-cadherin decreased in the experimental group of FoxM1 phosphorylation point mutants. In addition, the expression of PD-L1, which is known to be involved in immune evasion and increase the tumorigenic ability of cancer cells, was checked in each experimental group. As a result, high expression was observed in the FoxM1 phosphorylation point mutant experimental group (Figure 5D). Additionally, similar results were confirmed not only at the protein level but also at the mRNA level (Figure 5E). Therefore, it suggests that phosphorylation of FoxM1 enhances epithelial-mesenchymal transition, metastasis, and tumorigenic potential. On the other hand, it can be seen that the non-phosphorylated point mutants of the present invention, especially the S25A experimental group, are superior to the original FoxM1 experimental group in suppressing the metastatic ability and tumorigenic ability of cancer cells.
또한 FoxM1의 인산화 및 비인산화 점 돌연변이체 각 단백질이 발현되는 A549 세포에서 암세포의 세포사멸정도를 관찰하기 위하여 caspase-3 assay을 진행하여 caspase-3 효소의 활성을 측정하였다(도 5F). FoxM1의 인산화 및 비인산화 점 돌연변이체 각 단백질이 발현되는 A549 세포에 3μg/ml doxycycline를 48시간 동안 처리하여 각각의 돌연변이체의 과발현을 유도한 후에 세포를 lysis하여 caspase-3의 형광기질(Ac-DEVD-AMC)와 1시간 반응 후 전자분광분석기(spectramax M4 system)을 이용하여 형광값(Ex. 380nm/Emi. 430nm)을 측정하였다. 측정된 형광값은 caspase-3 활성으로 대조군의 측정값을 1이라고 할 때 각 실험군에서의 상대적 측정값의 정도를 산출하여 그래프로 표시하였다. 그 결과, FoxM1 비인산화 점 돌연변이체가 과발현된 세포에서 caspase-3 효소의 활성이 가장 높게 측정되었으며 반대로 FoxM1 인산화 점 돌연변이체가 과발현된 세포에서는 caspase-3 효소 활성이 가장 낮게 측정된 것을 관찰하였다. 이런 결과로 FoxM1 비인산화 점 돌연변이체의 과발현은 세포사멸성을 유도 및 증가시키는 것을 알 수 있었다.In addition, to observe the degree of apoptosis of cancer cells in A549 cells expressing each phosphorylated and non-phosphorylated point mutant protein of FoxM1, caspase-3 assay was performed to measure the activity of the caspase-3 enzyme (Figure 5F). Phosphorylated and non-phosphorylated point mutants of FoxM1 A549 cells expressing each protein were treated with 3 μg/ml doxycycline for 48 hours to induce overexpression of each mutant, and then the cells were lysed to produce the fluorescent substrate (Ac-3) of caspase-3. After reacting for 1 hour with DEVD-AMC), the fluorescence value (Ex. 380nm/Emi. 430nm) was measured using an electron spectrometer (spectramax M4 system). The measured fluorescence value was caspase-3 activity, and assuming that the control group's measurement value was 1, the relative measurement value in each experimental group was calculated and displayed on a graph. As a result, the highest caspase-3 enzyme activity was measured in cells overexpressing the non-phosphorylated FoxM1 point mutant, and conversely, the lowest caspase-3 enzyme activity was observed in cells overexpressing the FoxM1 phosphorylated point mutant. These results showed that overexpression of FoxM1 non-phosphorylated point mutant induced and increased apoptosis.
<실시예 1.9> FoxM1의 인산화 및 비인산화 점 돌연변이를 포함하는 단백질에 의한 대식세포의 분화능 평가 (도 6)<Example 1.9> Evaluation of differentiation capacity of macrophages by proteins containing phosphorylation and non-phosphorylation point mutations of FoxM1 (Figure 6)
동물조직에서 FoxM1 발현되는 암세포에서 PDL1 단밸질 발현과 CD274 mRNA 발현이 모두 증가된 것을 확인하였다(도 5D, 5E). 이러한 결과로 우리는 FoxM1 인산화 점 돌연변이체에서 종양면역회피가 일어날 가능성을 제기하고 종양면역회피에 영향을 줄 수 있는 종양미세환경에서의 면역세포의 분화에 미치는 영향을 연구하였다. 상위 결과로부터 우리는 FoxM1 인산화 점 돌연변이체(S25E)가 발현되는 암세포에서 종양미세환경에서의 대식세포 (Macrophage cell)의 분화(polarization)에 영향을 주는지 보고자 인간 단핵구 THP-1 세포와 FoxM1 점 돌연변이체가 과발현된 세포군들을 공배양한 후, THP-1 세포에서 M1, M2 마커를 관찰하였다. S25E가 발현되는 암세포 (4x104개/ml)와 THP-1 세포 (1.2x105개/ml)를 48h 동안 공배양 후 THP-1 세포에서 M1 마커인 INOS, IL12B의 mRNA 발현은 변화가 없으나 M2 마커인 IL10, CD163, CD206, TGFB1, VEGFA의 mRNA 발현은 모두 S25E에서 각각 2배이상 정도 증가함을 관찰하였다(도 6A). 그중 CD206, TGFB1, VEGFA는 M2d-종양 관련 대식세포(M2d-TAM)의 마커(JAYASINGAM, Sharmilla Devi, et al., Frontiers in Oncology (2020) 9: 1512.)로 알려져 있으며, FoxM1 S25E가 발현되는 A549 세포와 공배양한 THP-1 세포에서 M2d-종양 관련 대식세포의 마커들이 발현이 높게 관찰되었으므로 FoxM1 인산화 점 돌연변이체를 발현으로 인하여 THP-1 세포가 M2d-TAM로 분화됨을 알 수 있었다. In animal tissues, it was confirmed that both PDL1 protein expression and CD274 mRNA expression were increased in cancer cells expressing FoxM1 (Figures 5D and 5E). With these results, we raised the possibility that tumor immune evasion occurs in FoxM1 phosphorylation point mutants and studied the effect on the differentiation of immune cells in the tumor microenvironment, which may affect tumor immune evasion. From the top results, we aimed to report whether the FoxM1 phosphorylation point mutant (S25E) affects the polarization of macrophages in the tumor microenvironment in cancer cells expressing the FoxM1 phosphorylation point mutant (S25E) in human monocyte THP-1 cells and the FoxM1 point mutant. After co-culturing the overexpressed cell populations, M1 and M2 markers were observed in THP-1 cells. After co-culturing S25E-expressing cancer cells (4x10 4 cells/ml) and THP-1 cells (1.2x10 5 cells/ml) for 48 h, there was no change in the mRNA expression of M1 markers INOS and IL12B in THP-1 cells, but M2 It was observed that the mRNA expression of the markers IL10, CD163, CD206, TGFB1, and VEGFA all increased by more than 2-fold in S25E (Figure 6A). Among them, CD206, TGFB1, and VEGFA are known as markers of M2d-tumor-associated macrophages (M2d-TAM) (JAYASINGAM, Sharmilla Devi, et al., Frontiers in Oncology (2020) 9: 1512.), and FoxM1 S25E is expressed. Since high expression of markers of M2d-tumor-related macrophages was observed in THP-1 cells co-cultured with A549 cells, it was confirmed that THP-1 cells were differentiated into M2d-TAMs due to expression of FoxM1 phosphorylation point mutant.
또한 앞서 실험한 조건으로 인간 단핵구 THP1 세포와 FoxM1 점 돌연변이체가 과발현된 세포군들을 각각 48시간 공배양 후에 FoxM1 S25E가 발현되는 A549 세포에서 M2d 유도 인자인 IL4, IL6, IL10, VEGFA의 mRNA 발현수준을 분석한 결과, 이들 인자들이 각각 1.8배, 1.8배, 2.2배, 2.5배 2.5배 증가되었고, FoxM1 S25A 비인산화 변이체가 발현되는 A549 세포에서는 대조군과 비슷한 발현수준으로 현저히 감소됨을 관찰하였다(도 6B). FoxM1 변이체가 발현되는 A549 세포 (4x104개/ml)을 48h 동안 배양을 진행한 배지에서 TGFB1, VEGFA 단백질의 양을 ELISA로 측정한 결과, S25E에서 비교군 (Mock) 대비 각각 5배, 4배 정도로 현저히 증가되었으며 S25A에서는 비교군과 비슷한 수준으로 발현되므로 S25E에 비해 강하게 감소됨을 관찰하였다(도 6C). In addition, after co-culturing human monocyte THP1 cells and cell groups overexpressing FoxM1 point mutants for 48 hours under the previously tested conditions, the mRNA expression levels of M2d-inducing factors IL4, IL6, IL10, and VEGFA were analyzed in A549 cells expressing FoxM1 S25E. As a result, these factors were increased by 1.8-fold, 1.8-fold, 2.2-fold, 2.5-fold, and 2.5-fold, respectively, and in A549 cells expressing the non-phosphorylated FoxM1 S25A variant, it was observed that the expression level was significantly reduced to a level similar to that of the control group (Figure 6B). The amount of TGFB1 and VEGFA proteins was measured by ELISA in the medium in which A549 cells ( 4x10 cells/ml) expressing the FoxM1 variant were cultured for 48 h, and the results were 5-fold and 4-fold, respectively, in S25E compared to the comparison group (Mock). It was significantly increased, and in S25A, it was expressed at a similar level to the comparison group, so it was observed to be strongly reduced compared to S25E (Figure 6C).
다음으로는 FoxM1 인산화 점 돌연변이체(S25E)에 의해 M2 대식세포로의 분화에 직접적으로 영향을 미치는 지를 관찰하고자 FoxM1 발현 억제를 위한 발현억제시스템을 구축하였다. 먼저 FoxM1의 mRNA 발현을 억제하기 위하여 사람의 FoxM1 mRNA 서열 중 709-729 위치의 뉴클레오티드 서열을 각각 타겟으로 하는 shRNA를 만들고자 pLKO-puro.1 vector를 이용한 pLKO-puro.1-hFoxM1 플라스미드를 제작하였다. 사람의 FoxM1 shRNA 제작을 위한 타겟 서열로 하기의 서열이 가능하며 shRNA제작을 위한 프라이머로 하기의 서열을 지닌 올리고뉴클레오티드를 사용할 수 있다. Pubmed에서의 사람의 FoxM1 mRNA 유전자 접근번호는 NM_001243088.2 이며, 3507bp를 지니고 있다. shRNA 제작을 위한 primer 5'-ccgg-AGCAAGAGATGGAGGAAAAGG-ctcgag-CCTTTT CCTCCATCTCTTGCT-tttttg-3'(forward primer)와 5'-aattcaaaaa-AGCAAG AGATGGAGGAAAAGG-ctcgag-CCTTTTCCTCCATCTCTTGCT-3'(reverse primer)의 프라이머를 이용하였다. 그리고 제작한 각각의 FoxM1 shRNA 발현용 렌티바이러스를 정제 및 농축시킨 후 제작한 바이러스를 폐암세포인 FoxM1이 발현하는 A549 세포에 감염시켜 구축하였다. FoxM1 발현억제 한 폐암세포 (4x104개/ml)와 THP-1세포 (1.2x105개/ml)를 48h동안 공배양 한 후 M2 마커인 CD163, CD206, VEGFA의 mRNA의 발현를 관찰하였다. FoxM1 발현 억제시킨 S25E 세포주에서 M2 마커인 CD163, CD206, VEGFA의 mRNA의 발현이 현저히 감소되었다. 여기에 다시 FoxM1 변이체 S25E를 발현시킨 암세포와 공배양한 결과 THP1 세포에서 대조군(Mock_shCtrl) 대비 M2 마커인 CD163, CD206, VEGFA가 다시 모두 2배 이상 증가함을 확인하였다(도 6D). 즉, M2 대식세포로의 분화에 FoxM1 변이체 S25E가 직접적으로 영향을 미침을 의미한다. Next, an expression suppression system was constructed to suppress FoxM1 expression to observe whether the FoxM1 phosphorylation point mutant (S25E) directly affects differentiation into M2 macrophages. First, to suppress the mRNA expression of FoxM1, pLKO-puro.1-hFoxM1 plasmid was created using the pLKO-puro.1 vector to create shRNA targeting the nucleotide sequence at positions 709-729 of the human FoxM1 mRNA sequence. The following sequence can be used as a target sequence for producing human FoxM1 shRNA, and an oligonucleotide with the following sequence can be used as a primer for shRNA production. The human FoxM1 mRNA gene accession number in Pubmed is NM_001243088.2 and has 3507 bp. For shRNA production, the following primers were used: 5'-ccgg-AGCAAGAGATGGAGGAAAAGG-ctcgag-CCTTTT CCTCCATCTCTTGCT-tttttg-3' (forward primer) and 5'-aattcaaaaa-AGCAAG AGATGGAGGAAAAGG-ctcgag-CCTTTTCCTCCATCTCTTGCT-3' (reverse primer). After purifying and concentrating each of the produced lentiviruses for expressing FoxM1 shRNA, the produced viruses were constructed by infecting A549 cells expressing FoxM1, which are lung cancer cells. After co-culturing lung cancer cells (4x10 4 cells/ml) with suppressed FoxM1 expression and THP-1 cells (1.2x10 5 cells/ml) for 48 h, the expression of mRNA of M2 markers CD163, CD206, and VEGFA was observed. In the S25E cell line in which FoxM1 expression was suppressed, the mRNA expression of M2 markers CD163, CD206, and VEGFA was significantly reduced. As a result of co-culture with cancer cells expressing the FoxM1 mutant S25E, it was confirmed that the M2 markers CD163, CD206, and VEGFA in THP1 cells increased more than 2-fold compared to the control group (Mock_shCtrl) (Figure 6D). In other words, this means that FoxM1 variant S25E directly affects differentiation into M2 macrophages.
또한 마우스의 폐조직에서 광범위 대식세포 마커인 CD68와 종양관련대식세포 마커인 CD163 마커를 면역염색법을 이용하여 조직에서 분석한 결과, FoxM1 인산화 점 돌연변이체가 과발현된 세포를 주사한 마우스 폐조직에서 CD68, CD163 마커의 발현이 3배 정도 높게 관찰됨을 확인하였다(도 6E, 6F). 반면, 비인산화 점 돌연변이체에서는 CD68, CD163 마커의 발현은 Mock과 현저히 차이 없었고 인산화 점 돌연병체에 비해 낮음을 관찰하였다. 추가적으로 폐조직 용해액을 이용하여 면역블랏법으로 종양주변의 대식세포가 증가함을 관찰하고자 하였다. CD68와 CD163 단백질의 발현을 관찰한 결과, 인산화 점 돌연변이체(S25E)를 발현시키는 암세포를 주사한 마우스 폐조직에서 이들 종양 대식세포 마커의 발현이 현저히 높은 반면, 비인산화 점 돌연변이체(S25A)를 발현시킨 세포를 주사한 마우스 폐조직에서 이들 종양 대식세포 마커의 발현이 대조군보다 낮음을 관찰할 수 있었다(도 6G). 본 연구결과를 통하여 비인산화 점 돌연변이체가 종양관련 대식세포로의 분화를 억제하는 효과를 관찰할 수 있었다.In addition, as a result of analyzing CD68, a broad macrophage marker, and CD163, a tumor-related macrophage marker, in mouse lung tissue using immunostaining, the results showed that CD68, It was confirmed that the expression of the CD163 marker was observed to be about 3 times higher (Figures 6E, 6F). On the other hand, the expression of CD68 and CD163 markers in the non-phosphorylated point mutant was not significantly different from the mock and was observed to be lower than that in the phosphorylated point mutant. Additionally, we attempted to observe the increase in macrophages around the tumor by immunoblotting using lung tissue lysate. As a result of observing the expression of CD68 and CD163 proteins, the expression of these tumor macrophage markers was significantly higher in mouse lung tissue injected with cancer cells expressing the phosphorylated point mutant (S25E), whereas the expression of these tumor macrophage markers was significantly higher in mouse lung tissue injected with cancer cells expressing the non-phosphorylated point mutant (S25A). In mouse lung tissue injected with the expressed cells, the expression of these tumor macrophage markers was observed to be lower than that in the control group (Figure 6G). Through the results of this study, we were able to observe the effect of non-phosphorylated point mutants on suppressing differentiation into tumor-related macrophages.
<실시예 1.10> FoxM1의 인산화 및 비인산화 점 돌연변이를 포함하는 단백질에 의한 종양면역회피능 평가 (도 7)<Example 1.10> Evaluation of tumor immune evasion ability by proteins containing phosphorylation and non-phosphorylation point mutations of FoxM1 (Figure 7)
FoxM1의 인산화 및 비인산화 점 돌연변이가 발현되는 폐암(A549)세포와 단핵세포인 THP-1 세포를 공배양 후에 폐암세포의 생존율에 영향을 주는지를 관찰하고자 세포의 생존율을 측정하였다. A549 세포(4x104개/ml) : THP-1세포의 비율을 1:0, 1:2, 1:4, 1:6로 변화시킨 후 48h 동안 공배양 후에 THP-1 세포를 씻겨 버리고 바닥에 붙어 있는 A549 세포의 생존율을 MTT assay로 측정하였다.After co-culturing lung cancer (A549) cells expressing phosphorylation and non-phosphorylation point mutations in FoxM1 and THP-1 cells, which are mononuclear cells, the survival rate of the cells was measured to observe whether it affected the survival rate of lung cancer cells. A549 cells ( 4x10 cells/ml): Change the ratio of THP-1 cells to 1:0, 1:2, 1:4, 1:6, and co-culture for 48 h. Then, wash the THP-1 cells and place them on the bottom. The survival rate of attached A549 cells was measured by MTT assay.
측정결과, FoxM1 인산화 점 돌연변이체를 발현하는 A549 (A549S25E)세포의 생존율이 THP-1 비율에 따라 점차 증가함으로 관찰할 수 있었고, A549 세포: THP-1세포의 비율 1:6에서 생존율이 제일 높음을 관찰할 수 있었다(도 7A). 반면, FoxM1 비인산화 점 돌연변이체를 발현하는 A549 (A549S25A)세포의 생존율은 대조군과 비교하여 변화가 거의 없었다.As a result of the measurement, it was observed that the survival rate of A549 (A549 S25E ) cells expressing the FoxM1 phosphorylation point mutant gradually increased depending on the THP-1 ratio, and the survival rate was highest at the ratio of A549 cells: THP-1 cells of 1:6. high was observed (Figure 7A). On the other hand, the survival rate of A549 (A549 S25A ) cells expressing the non-phosphorylated FoxM1 point mutant showed little change compared to the control group.
FoxM1 인산화 점 돌연변이체가 발현되는 세포와 THP-1 세포에서 고형암 면역회피에 관여하는 PD-1과 PD-L1의 mRNA 발현수준을 검토하기 위하여 A549S25E세포와 48h 동안 공배양한 THP-1 단핵세포에서 면역회피 인자인 PD-1 mRNA (CD279) 발현을 qRT-PCR로 측정하였다(도 7B). 인산화 점 돌연변이체 발현 A549S25E 세포에서 면역회피 인자 PD-L1의 mRNA (CD274)발현이 대조군 대비 2배 증가함을 관찰하였다. To examine the mRNA expression levels of PD-1 and PD-L1, which are involved in solid tumor immune evasion, in THP-1 cells and cells expressing FoxM1 phosphorylation point mutants, THP-1 mononuclear cells co-cultured with A549 S25E cells for 48 h. Expression of PD-1 mRNA (CD279), an immune evasion factor, was measured by qRT-PCR (Figure 7B). It was observed that the mRNA (CD274) expression of the immune evasion factor PD-L1 (CD274) in A549S25E cells expressing the phosphorylation point mutant increased two-fold compared to the control group.
또한, FoxM1 S25E 인산화 점 돌연변이체를 발현하는 A549 (A549S25E)세포와 T 세포인 Jurkat 세포과 공배양을 통하여 T 세포의 종양면역능력에 미치는 효과를 관찰하고자 하였다(도 7D). Jurkat 세포과 48h 공배양한 인산화 변이체 발현 A549S25E 세포(4x104개/ml)의 생존율은 Jurkat 세포의 비율을 1:0, 1:2, 1:4, 1:6 늘리수록 1.2배, 1.3배, 1.5배로 점차 증가되는 것이 관찰된 반면 A549S25A세포의 생존율은 원형이나 인산화 변이체 발현 A549 세포 대비 현저히 억제됨을 관찰할 수 있었다(도 7D). In addition, we attempted to observe the effect on the tumor immune capacity of T cells by co-culturing A549 (A549 S25E ) cells expressing the FoxM1 S25E phosphorylation point mutant with Jurkat cells, which are T cells (Figure 7D). The survival rate of A549 S25E cells ( 4x10 cells/ml) expressing the phosphorylated mutant co-cultured with Jurkat cells for 48 h increased by 1.2-fold, 1.3-fold, and 1.3-fold as the ratio of Jurkat cells was increased to 1:0, 1:2, 1:4, and 1:6. While a gradual increase of 1.5-fold was observed, the survival rate of A549 S25A cells was observed to be significantly suppressed compared to A549 cells expressing the original or phosphorylated variant (Figure 7D).
이는 A549S25E 세포가 T 세포의 성질에도 영향을 미칠 것으로 보여, A549S25E 세포에 의한 tumor-infiltrating T lymphocytes (TILs)로의 분화를 검토하고자 Jurkat 세포 (1.2x105개)와 A549S25E 세포 (4x104개)로 48h 동안 공배양 후 TILs에서 발현되는 CD25와 CD29의 발현 수준을 qRT-PCR로 분석하였다. 그 결과, A549S25E 세포와 공배양된 Jurkat 세포에서 CD25와 CD29의 발현이 현저히 3배 이상 증가됨을 관찰하였다. 하지만 A549S25A 세포와 공배양된 Jurkat 세포에서는 원형이나 인산화 점 돌연변이체 발현 A549 세포와 공배양된 Jurkat 세포 대비, 현저히 억제하는 효과가 있음을 보여주었다(도 7E). 앞서 실험 조건에서 A549S25E 세포에서 TILs 분화를 유도하는 IL6와 IL1A 및 종양면역회피인자인 CD274의 mRNA 발현이 대조군 대비 양적으로 각각 3.5배, 4.5배, 5배 증가가 있음을 관찰한 반면 A549S25A 세포에서 이들 인자의 발현이 현저히 낮음을 관찰하여 억제하는 효과가 있음을 보여주었다(도 7F). 추가적으로 종양미세환경에서 TAMs나 TILs에 의해 종양면역회피 반응에 A549S25A 세포가 억제효과를 있음을 FoxM1 인산화 및 비인산화 점 돌연변이체를 발현하는 A549 세포와 THP-1세포, Jurkat 세포와의 효과를 보고자 각각 비율을 1:0:0, 1:6:0, 1:0:6, 1:6:6 으로 48h 동안 공배양 후 생존율 변화를MTT assay 분석을 통하여 관찰하였다. 그 결과, FoxM1 인산화 점 돌연변이체(S25E)에서 현저히 증가되었으며 비인산화 점 돌연변이체(S25A)에서는 감소되는 것을 관찰할 수 있었다(도 7G). 즉, 종양미세환경에서 TAMs나 TILs에 의해 종양면역회피 반응에 비인산화 점 돌연변이체를 발현하는 세포가 억제효과를 있음을 보여준다. 그러므로 본 발명은 FoxM1의 비인산화 점 돌연변이체에 의한 종양 면역회피반응 억제 효과를 제공한다. This suggests that A549 S25E cells may also affect the properties of T cells. To examine the differentiation of A549 S25E cells into tumor-infiltrating T lymphocytes (TILs), Jurkat cells (1.2x10 5 cells) and A549 S25E cells (4x10 4 cells) were used. ), the expression levels of CD25 and CD29 expressed in TILs were analyzed by qRT-PCR after co-culture for 48 h. As a result, it was observed that the expression of CD25 and CD29 was significantly increased by more than 3-fold in Jurkat cells co-cultured with A549 S25E cells. However, Jurkat cells co-cultured with A549 S25A cells showed a significant inhibitory effect compared to Jurkat cells co-cultured with A549 cells expressing the original or phosphorylated point mutant (Figure 7E). In the previous experimental conditions, it was observed that the mRNA expression of IL6 and IL1A, which induce TIL differentiation, and CD274, a tumor immune evasion factor, in A549 S25E cells quantitatively increased by 3.5-fold, 4.5-fold, and 5-fold, respectively, compared to the control group. It was observed that the expression of these factors was significantly low, showing that there was an inhibitory effect (Figure 7F). Additionally, we aimed to report the inhibitory effect of A549 S25A cells on the tumor immune evasion response by TAMs or TILs in the tumor microenvironment with A549 cells expressing phosphorylated and non-phosphorylated FoxM1 point mutants, THP-1 cells, and Jurkat cells. After co-culturing for 48 h at the ratios of 1:0:0, 1:6:0, 1:0:6, and 1:6:6, changes in survival rate were observed through MTT assay analysis. As a result, it was observed that it was significantly increased in the FoxM1 phosphorylated point mutant (S25E) and decreased in the non-phosphorylated point mutant (S25A) (Figure 7G). In other words, it shows that cells expressing non-phosphorylated point mutants have an inhibitory effect on the tumor immune evasion response caused by TAMs or TILs in the tumor microenvironment. Therefore, the present invention provides the effect of suppressing tumor immune evasion response by a non-phosphorylated point mutant of FoxM1.
<실시예 1.11> FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드에 의한 암세포 사멸성 및 암세포 이동성 억제에 대한 효능 평가 (도 8) <Example 1.11> Evaluation of efficacy on inhibition of cancer cell apoptosis and cancer cell mobility by peptide containing non-phosphorylation point mutation of FoxM1 (FIG. 8)
본 발명자들은 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드에 의한 암세포 사멸성 및 암세포 이동성에 관한 억제효과에 대한 효능을 평가하기 위하여 FoxM1의 비인산화 점 돌연변이를 포함하는 3종류의 QNAPAETSEE(set #1, 서열번호 4), PAETSEEEPK(set #2, 서열번호 2), LPVQNAPAET(set #3, 서열번호 5)의 아미노산에 세포내에 침투시키기 위한 CPP(Cell-Penetrating Peptide)의 한 종류인 TAT(YGRKKRRQRRR)를 붙여서 펩티드 합성을 하였다(도 8A). 먼저 제작한 3개의 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드에 암세포 사멸성에 관한 효능을 평가하기 위해서 caspase-3 assay를 통해서 caspase-3 효소의 활성을 측정하여 암세포의 세포 사멸성을 관찰하였다(도 8B). A549 폐암세포에 각각 3종류의 5μM 펩티드를 48시간 동안 처리후에 펩티드를 처리한 각각의 세포를 lysis하여 caspase-3의 형광기질(Ac-DEVD-AMC)와 2시간 반응 후 전자분광분석기(spectramax M4 system)을 이용하여 형광값(Ex. 380nm/Emi. 430nm)을 측정하였다. 측정된 형광값은 caspase-3 활성으로 상대 형광 단위(relative fluorescence units, RFU)로 표시하여 세포사멸정도를 측정하였다. 그 결과, 3종류의 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드는 모두 암세포의 세포사멸성이 관찰되었으며, 특히 펩티드#1, #3에서 그 효능이 높게 나타나는 것을 관찰할 수 있었다. The present inventors used three types of QNAPAETSEE (set #1, set #1, TAT (YGRKKRRQRRR), a type of CPP (Cell-Penetrating Peptide), is used to infiltrate the amino acids of SEQ ID NO: 4), PAETSEEEPK (set #2, SEQ ID NO: 2), and LPVQNAPAET (set #3, SEQ ID NO: 5) into cells. Peptide synthesis was performed (Figure 8A). In order to evaluate the efficacy of the peptides containing the three non-phosphorylation point mutations of FoxM1 produced first on cancer cell apoptosis, the activity of the caspase-3 enzyme was measured through a caspase-3 assay and the apoptosis of cancer cells was observed (Figure 8B). After treating A549 lung cancer cells with three types of 5 μM peptides for 48 hours, each cell treated with the peptides was lysed and reacted with the fluorescent substrate of caspase-3 (Ac-DEVD-AMC) for 2 hours, then analyzed using an electron spectrometer (spectramax M4). The fluorescence value (Ex. 380nm/Emi. 430nm) was measured using the system. The measured fluorescence value was expressed in relative fluorescence units (RFU) as caspase-3 activity to measure the degree of cell death. As a result, all 3 types of peptides containing non-phosphorylation point mutations in FoxM1 were observed to cause apoptosis in cancer cells, and peptides #1 and #3 showed particularly high efficacy.
또한 제작한 3개의 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드에 암세포 이동성에 미치는 효과 및 각각의 펩티드에 관한 효능을 평가하기 위해서 migration assay를 통해서 세포 이동성을 측정하였다(도 8C). 구체적으로, FoxM1 인산화 점 돌연변이체 단백질을 발현시키는 폐암세포 A549을 5 x 104 개의 세포를 8.0 μm, 24 well insert에 분주하고 24 well plate에 10% 혈청(FBS)이 포함된 배지를 분주한 뒤 inset를 넣어 주었다. 3μg/ml doxycycline이 포함된 RPMI 1640(10% FBS)를 0.5 ml 분주하였으며, 각각 3종류의 5 μM FoxM1 비인산화 점 돌연변이 펩티드를 동시에 처리하였다. 세포 분주 48시간 후, 4% 파라포름알데히드(paraformaldehyde) 500 ㎕를 분주하고 1XPBS로 3회 세척하여 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도(intensity)를 Odyssey infrared imaging system을 이용하여 측정하였다. 대조군의 염색된 정도(intensity)를 1이라고 할 때 각 실험군에서의 상대적 염색된 정도를 산출하여 그래프를 표시하였다. 연구 결과, FoxM1 인산화 점 돌연변이체 과발현군에서 염색강도(intensity) 수치가 높게 관찰되었으며 대조군 대비 5배까지 증가하였으며, 각각의 FoxM1 비인산화 점 돌연변이체 펩티드 처리에 의해서 염색강도가 20-40% 정도로 수치로 감소한 것을 관찰할 수 있었다(도 8C). 따라서 3개의 FoxM1 비인산화 점 돌연변이체 펩티드 처리에 의해서 A549 폐암세포의 대조군에서 뿐만 아니라 FoxM1 인산화에 의해 증가된 세포이동성도 크게 억제되는 것을 관찰하였다. 특히 펩티드#1과 펩티드#3에서 암세포 이동성에 대한 억제 효과가 좋은 것으로 관찰되었다.In addition, in order to evaluate the effect of the three produced peptides containing non-phosphorylation point mutations of FoxM1 on cancer cell mobility and the efficacy of each peptide, cell mobility was measured through a migration assay (Figure 8C). Specifically, 5 x 10 4 cells of lung cancer cell A549 expressing FoxM1 phosphorylated point mutant protein were dispensed into 8.0 μm, 24 well inserts, and medium containing 10% serum (FBS) was dispensed into the 24 well plate. I added an inset. 0.5 ml of RPMI 1640 (10% FBS) containing 3 μg/ml doxycycline was dispensed, and three types of 5 μM FoxM1 non-phosphorylated point mutant peptides were simultaneously treated. 48 hours after cell distribution, 500 ㎕ of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the intensity of staining was measured using the Odyssey infrared imaging system. Assuming that the intensity of staining in the control group was 1, the relative staining intensity in each experimental group was calculated and displayed on a graph. As a result of the study, high staining intensity was observed in the group overexpressing the FoxM1 phosphorylated point mutant, increasing up to 5 times compared to the control group, and the staining intensity increased to about 20-40% by treatment with each non-phosphorylated point mutant peptide of FoxM1. A decrease was observed (Figure 8C). Therefore, it was observed that treatment with three non-phosphorylated FoxM1 point mutant peptides significantly inhibited cell migration increased by FoxM1 phosphorylation as well as in control A549 lung cancer cells. In particular, peptide #1 and peptide #3 were observed to have a good inhibitory effect on cancer cell mobility.
이상의 결과로 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드가 폐암세포의 사멸성 및 암세포 이동성 억제 효과가 있음을 시사하였다.The above results suggested that the peptide containing the non-phosphorylated point mutation of FoxM1 had an effect on suppressing the apoptosis and cancer cell mobility of lung cancer cells.
<실시예 1.12> FoxM1 발현에 따른 암종별 환자생존율에 미치는 영향 평가 (도 9)<Example 1.12> Evaluation of the impact of FoxM1 expression on patient survival rate by cancer type (Figure 9)
다양한 암종에서 FoxM1의 발현량이 임상적으로 의미가 있는지 확인하기 위해서 암종별 환자에서 FoxM1의 발현량에 따른 환자의 생존율을 빅테이터 분석을 통해서 확인하였다(도 9A). Kaplan Meier 플로터(https://kmplot.com/analysis)를 이용하여 다양한 종양 유형에서 FoxM1 발현과 환자의 생존 사이의 상관관계를 평가하였다. 먼저 12개의 암종별로 FoxM1의 발현과 생존율을 분석한 결과, FoxM1 발현이 높은 환자의 생존율이 FoxM1 발현이 낮은 환자의 생존율에 비해 현저히 낮은 것을 확인할 수 있었다. 특히 12개의 암종 중 폐암(n=504, HR=2, log rank P=2.8e-05), 유방암(n=947, HR=2.01, log rank P=0.0089), 신장암(n=530, HR=2.81, log rank P=1.9e-12), 간암(n=370, HR=2.07, log rank P=5.1e-05), 갑상선암(n=353, HR=5.24, log rank P=6.9e-05), 췌장암(n=69, HR=3.7, log rank P=0.022), 식도암(n=19, HR=8.35, log rank P=0.03), 자궁내막암(n=542, HR=1.7, log rank P=0.011), 육종암(n=152, HR=2.51, log rank P=0.00016) 그리고 갈색세포종 부신 종양(n=178, HR=6.98, log rank P=0.041)에서 FoxM1 발현이 높은 환자의 생존율이 낮게 관찰된 결과뿐만 아니라 유의성(log rank P < 0.05)이 있는 결과도 관찰할 수 있었다. 또한 고환생식세포종양(n=105, HR=2.76, log rank P=0.051) 및 자궁경부 편평상피세포암종(n=174, HR=1.89, log rank P=0.098)에서도 FoxM1 발현이 높은 환자 생존기간의 상대 위험비(Harzard ratio, HR)가 1.2이상으로 높게 관찰되었다.In order to confirm whether the expression level of FoxM1 in various cancer types is clinically meaningful, the survival rate of patients according to the expression level of FoxM1 in patients of each cancer type was confirmed through big data analysis (Figure 9A). The correlation between FoxM1 expression and patient survival in various tumor types was evaluated using Kaplan Meier plotter (https://kmplot.com/analysis). First, as a result of analyzing the expression and survival rate of FoxM1 for each 12 carcinomas, it was confirmed that the survival rate of patients with high FoxM1 expression was significantly lower than the survival rate of patients with low FoxM1 expression. In particular, among the 12 carcinomas, lung cancer (n=504, HR=2, log rank P=2.8e-05), breast cancer (n=947, HR=2.01, log rank P=0.0089), and kidney cancer (n=530, HR) =2.81, log rank P=1.9e-12), liver cancer (n=370, HR=2.07, log rank P=5.1e-05), thyroid cancer (n=353, HR=5.24, log rank P=6.9e- 05), pancreatic cancer (n=69, HR=3.7, log rank P=0.022), esophageal cancer (n=19, HR=8.35, log rank P=0.03), endometrial cancer (n=542, HR=1.7, log rank P=0.011), sarcoma (n=152, HR=2.51, log rank P=0.00016), and pheochromocytoma adrenal tumor (n=178, HR=6.98, log rank P=0.041) in patients with high FoxM1 expression. Not only were low survival rates observed, but also results with significance (log rank P < 0.05) were observed. Additionally, the survival time of patients with high FoxM1 expression was observed in testicular germ cell tumor (n=105, HR=2.76, log rank P=0.051) and cervical squamous cell carcinoma (n=174, HR=1.89, log rank P=0.098). The relative risk ratio (HR) was observed to be high, over 1.2.
이러한 결과는 FoxM1 발현이 여러 암종에서 환자의 생존율에 영향을 주는 것을 시사하였다.These results suggested that FoxM1 expression affects patient survival in several carcinomas.
또한 본 발명자는 여러 암종에서 앞서 제작한 3개의 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드의 암세포 사멸성에 관한 효능을 평가하고자 하였다. 도 8B와 같은 방법으로 caspase-3 assay를 통해서 caspase-3 효소의 활성을 측정하여 여러 암세포의 세포 사멸성을 관찰하였다(도 9B). 자궁경부암세포(HeLa)와 간암세포(Hep3B)에 각각 3종류의 5μM 펩티드를 48시간 동안 처리 후에 펩티드를 처리한 각각의 세포를 lysis하여 caspase-3의 형광기질(Ac-DEVD-AMC)과 2시간 반응 후 전자분광분석기(spectramax M4 system)를 이용하여 형광 값(Ex. 380nm/Emi. 430nm)을 측정하였다. 측정된 형광 값은 caspase-3 활성으로 상대 형광 단위(relative fluorescence units, RFU)로 표시하여 세포사멸 정도를 측정하였다. 그 결과, 자궁경부암 세포(HeLa)와 간암세포(Hep3B)에서 3종류의 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드는 모두 암세포의 세포사멸성이 관찰되었으며, 특히 펩티드#1에서 그 효능이 높게 나타나는 것을 관찰할 수 있었다. 또한 펩티드의 세포사멸성에 관한 효능이 자궁경부암에서 보다 간암에서 더 큰 것을 관찰할 수 있었다.In addition, the present inventors sought to evaluate the efficacy of peptides containing three non-phosphorylation point mutations of FoxM1 previously prepared in various cancer types on cancer cell killing. The activity of the caspase-3 enzyme was measured through a caspase-3 assay in the same manner as shown in Figure 8B, and the apoptosis of several cancer cells was observed (Figure 9B). Cervical cancer cells (HeLa) and liver cancer cells (Hep3B) were treated with three types of 5 μM peptides each for 48 hours, and each cell treated with the peptides was lysed to produce caspase-3 fluorescent substrate (Ac-DEVD-AMC) and 2 After the time reaction, the fluorescence value (Ex. 380nm/Emi. 430nm) was measured using an electron spectrometer (spectramax M4 system). The measured fluorescence value was expressed in relative fluorescence units (RFU) as caspase-3 activity to measure the degree of apoptosis. As a result, in cervical cancer cells (HeLa) and liver cancer cells (Hep3B), all 3 types of peptides containing non-phosphorylation point mutations of FoxM1 were observed to cause cancer cell apoptosis, and peptide #1 showed particularly high efficacy. could be observed. In addition, it was observed that the efficacy of the peptide on apoptosis was greater in liver cancer than in cervical cancer.
따라서 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드는 폐암뿐만 아니라 여러 암종에서 세포사멸성을 가질 수 있음을 시사하였다.Therefore, it was suggested that peptides containing non-phosphorylated point mutations in FoxM1 may have apoptotic properties not only in lung cancer but also in various carcinomas.
<실시예 1.13> FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드에 의한 암세포 이동성 및 대식세포의 분화 억제효과 평가 (도 10) <Example 1.13> Evaluation of the inhibitory effect on cancer cell mobility and macrophage differentiation by a peptide containing a non-phosphorylated point mutation of FoxM1 (FIG. 10)
본 발명자들은 FoxM1의 비인산화 점 돌연변이를 포함하는 펩티드에 의한 전이성 환경에서 암세포 이동성 및 대식세포의 분화 억제효과를 평가하기 위하여 가장 효능이 높게 평가된 FoxM1 비인산화 점 돌연변이 펩티드 #1를 이용하여 실험을 진행하였다. 먼저 FoxM1 비인산화 점 돌연변이 펩티드의 세포내 침투여부를 알아보기 위해서 펩티드의 C 말단쪽에 FITC 형광물질을 붙여서 제작하였다(도 10A). The present inventors conducted an experiment using FoxM1 non-phosphorylated point mutant peptide #1, which was evaluated as the most effective, to evaluate the inhibitory effect on cancer cell mobility and macrophage differentiation in the metastatic environment caused by a peptide containing a non-phosphorylated point mutation of FoxM1. proceeded. First, to determine whether the FoxM1 non-phosphorylated point mutant peptide penetrates into cells, a FITC fluorescent substance was attached to the C terminus of the peptide to prepare it (Figure 10A).
폐암 세포인 A549에 5 μM FoxM1 비인산화 점 돌연변이 펩티드를 24시간 처리 후 처리된 펩티드가 세포내에 투과되는 지를 형광현미경으로 FITC 형광을 관찰하였다. 도 10B에서 같이, 펩티드 처리 24시간 후에 펩티드가 세포내에 잘 투과됨을 관찰하였다. 또한 5ng/ml TGF-β에 의해 유도된 암전이 조건에서 세포내로 투과된 펩티드의 처리가 상피간엽이행 마커(EMT marker)의 mRNA 발현 변화를 시간 중합효소 연쇄반응(Real-time PCR)을 통해서 관찰하였다. 먼저 TGF-β를 48시간 처리한 군에서 간엽이행 마커인 CDH2와 vimentin mRNA 발현이 증가하였으나 펩티드 처리에 의해서 발현이 감소하는 것을 관찰하였다. 반대로 TGF-β 처리군에서 상피형질 마커인 CDH1의 mRNA 발현 감소가 펩티드 처리에 의해서 증가되는 것을 관찰하였다(도 10C). After treating lung cancer cells A549 with 5 μM FoxM1 non-phosphorylated point mutant peptide for 24 hours, FITC fluorescence was observed under a fluorescence microscope to determine whether the treated peptide penetrated the cells. As shown in Figure 10B, it was observed that the peptide penetrated well into the cells 24 hours after treatment. In addition, under cancer metastasis conditions induced by 5ng/ml TGF-β, changes in the mRNA expression of epithelial-mesenchymal transition markers (EMT markers) were observed through real-time PCR when treated with peptides that penetrated into cells. did. First, in the group treated with TGF-β for 48 hours, the expression of mesenchymal transition markers CDH2 and vimentin mRNA increased, but the expression was observed to decrease due to peptide treatment. Conversely, in the TGF-β treatment group, a decrease in mRNA expression of CDH1, an epithelial trait marker, was observed to be increased by peptide treatment (Figure 10C).
FoxM1 비인산화 점 돌연변이 펩티드 처리가 TGF-β가 처리된 전이환경에서 암세포의 이동성에 미치는 효과를 관찰하기 위해 세포이동성 실험 (migration assay)을 실시하였다.A cell migration assay was performed to observe the effect of FoxM1 non-phosphorylated point mutant peptide treatment on the mobility of cancer cells in a metastatic environment treated with TGF-β.
구체적으로, 폐암세포 A549를 5x104 개의 세포를 8.0 μm, 24 well insert에 분주하고 24 well plate에 10% 혈청 (FBS)이 포함된 배지를 분주한 뒤 inset를 넣어 주었다. 양성 대조군의 경우, 5 ng/ml TGF-β가 포함된 RPMI 1640(10% FBS)를 0.5 ml 분주하여 사용하였으며, 5 μM 비인산화 점 돌연변이 펩티드를 동시에 처리하였다. 세포 분주 48시간 후, 4% 파라포름알데히드(paraformaldehyde) 500 ㎕를 분주하고 1XPBS로 3회 세척하여 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도(intensity)를 Odyssey infrared imaging system을 이용하여 측정하였다. 대조군의 염색된 정도(intensity)를 1이라고 할 때 각 실험군에서의 상대적 염색된 정도를 산출하여 그래프를 표시하였다.Specifically, 5x10 4 cells of lung cancer cell A549 were dispensed into an 8.0 μm, 24 well insert, medium containing 10% serum (FBS) was dispensed into the 24 well plate, and the inset was added. For the positive control, 0.5 ml of RPMI 1640 (10% FBS) containing 5 ng/ml TGF-β was used, and 5 μM non-phosphorylated point mutant peptide was simultaneously treated. 48 hours after cell distribution, 500 ㎕ of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the intensity of staining was measured using the Odyssey infrared imaging system. Assuming that the intensity of staining in the control group was 1, the relative staining intensity in each experimental group was calculated and displayed on a graph.
연구 결과, TGF-β 처리군에서 염색강도(intensity) 수치가 높게 관찰되었으며 대조군 대비 4배까지 증가하였으며, FoxM1 비인산화 점 돌연변이체 펩티드 처리에 의해서 염색강도가 대조군과 유사한 수치로 감소한 것을 관찰할 수 있었다(도 10D). 따라서 FoxM1 비인산화 점 돌연변이체 펩티드 처리에 의해서 TGF-β 처리에 의해 유도되는 세포이동성에 대한 강력한 억제효과가 있음을 알 수 있었다. As a result of the study, the staining intensity value was observed to be high in the TGF-β treatment group and increased up to 4 times compared to the control group. By treatment with FoxM1 non-phosphorylated point mutant peptide, the staining intensity was reduced to a value similar to that of the control group. There was (Figure 10D). Therefore, it was found that treatment with FoxM1 non-phosphorylated point mutant peptide had a strong inhibitory effect on cell migration induced by TGF-β treatment.
이상의 결과는 TGF-β 처리에 의해 유도되는 전이성 환경에서 FoxM1 비인산화 점 돌연변이체 펩티드는 전이성 억제 효과가 있음을 시사하였다.The above results suggested that FoxM1 non-phosphorylated point mutant peptide had a metastatic inhibitory effect in the metastatic environment induced by TGF-β treatment.
본 발명자는 FoxM1 인산화 점 돌연변이체 단백질을 과발현시키는 폐암 세포 A549에 FoxM1 비인산화 점 돌연변이 펩티드 처리가 중간엽이행 마커 mRNA 변화를 관찰하기 위해서 상피간엽이행 마커(EMT marker)의 mRNA 발현 변화를 시간 중합효소 연쇄반응(Real-time PCR)을 통해서 관찰하였다. FoxM1 인산화 점 돌연변이체의 과발현 시스템이 구축된 세포에 3μg/ml doxycycline 24시간 처리하여 과발현을 유도 후 FoxM1 비인산화 점 돌연변이 펩티드 처리를 24시간 처리하여 FoxM1 과발현 정도 및 상피간엽이행 마커(EMT marker)의 mRNA 발현량으로 확인하였다. 도 10E에서와 같이, FoxM1의 과발현은 잘 되고 있는 것을 관찰하였으며, 펩티드 처리에 의해서 FoxM1의 과발현에 의해서 증가된 간엽이행 마커인 CDH2와 vimentin mRNA 발현이 감소하였으며, 반대로 상피형질 마커인 CDH1의 mRNA 발현 감소가 펩티드 처리에 의해서 증가되는 것을 관찰하였다(도 10E). The present inventors investigated changes in the mRNA expression of epithelial-mesenchymal transition markers (EMT markers) by treating lung cancer cells A549, which overexpress FoxM1 phosphorylated point mutant proteins, with FoxM1 non-phosphorylated point mutant peptides. Observation was made through chain reaction (real-time PCR). Cells constructed with an overexpression system for FoxM1 phosphorylated point mutants were treated with 3 μg/ml doxycycline for 24 hours to induce overexpression, and then treated with FoxM1 non-phosphorylated point mutant peptide for 24 hours to determine the degree of FoxM1 overexpression and epithelial-mesenchymal transition marker (EMT marker). This was confirmed by the mRNA expression level. As shown in Figure 10E, it was observed that the overexpression of FoxM1 was working well. By peptide treatment, the mRNA expression of CDH2 and vimentin, a mesenchymal transition marker, which was increased by the overexpression of FoxM1, decreased, and conversely, the mRNA expression of CDH1, an epithelial trait marker, was decreased. We observed that the reduction was increased by peptide treatment (Figure 10E).
또한 A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 FoxM1 비인산화 점 돌연변이체 펩티드(5μM FoxM1-S25A peptide)처리에 의해 암세포의 이동성의 변화를 관찰하였다. In addition, changes in the mobility of cancer cells were observed by treatment with non-phosphorylated FoxM1 point mutant peptide (5 μM FoxM1-S25A peptide) under conditions of overexpression of a phosphorylated point mutant of FoxM1 in A549 cells.
구체적으로, FoxM1 인산화 점 돌연변이체 단백질을 발현시키는 폐암세포 A549을 5 x 104 개의 세포를 8.0 μm, 24 well insert에 분주하고 24 well plate에 10% 혈청(FBS)이 포함된 배지를 분주한 뒤 inset를 넣어 주었다. 3ug/ml doxycycline이 포함된 RPMI 1640(10% FBS)를 0.5 ml 분주하였으며, 5 uM 비인산화 점돌연변이 펩티드를 동시에 처리하였다. 세포 분주 48시간 후, 4% 파라포름알데히드(paraformaldehyde) 500 ㎕를 분주하고 1XPBS로 3회 세척하여 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도(intensity)를 Odyssey infrared imaging system을 이용하여 측정하였다. 대조군의 염색된 정도(intensity)를 1이라고 할 때 각 실험군에서의 상대적 염색된 정도를 산출하여 그래프를 표시하였다. Specifically, 5 was put in. 0.5 ml of RPMI 1640 (10% FBS) containing 3ug/ml doxycycline was dispensed, and 5 uM non-phosphorylated point mutant peptide was simultaneously treated. 48 hours after cell distribution, 500 ㎕ of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the intensity of staining was measured using the Odyssey infrared imaging system. Assuming that the intensity of staining in the control group was 1, the relative staining intensity in each experimental group was calculated and displayed on a graph.
연구 결과, FoxM1 인산화 점 돌연변이체 과발현군에서 염색강도(intensity) 수치가 높게 관찰되었으며 대조군 대비 5배까지 증가하였으며, FoxM1 비인산화 점 돌연변이체 펩티드 처리에 의해서 염색강도가 50% 정도로 수치로 감소한 것을 관찰할 수 있었다(도 10F). 따라서 FoxM1 비인산화 점 돌연변이체 펩티드 처리에 의해서 FoxM1 인산화에 의해 증가된 세포이동성이 억제되는 것을 관찰하였다.As a result of the study, the staining intensity was observed to be high in the group overexpressing the FoxM1 phosphorylated point mutant, increasing up to 5 times compared to the control group, and the staining intensity was observed to decrease to about 50% by treatment with the FoxM1 non-phosphorylated point mutant peptide. It was possible (Figure 10F). Therefore, it was observed that the increased cell migration caused by FoxM1 phosphorylation was suppressed by treatment with FoxM1 non-phosphorylated point mutant peptide.
마지막으로, A549 세포에 FoxM1의 인산화 점 돌연변이체의 과발현 조건에서 FoxM1 비인산화 점 돌연변이체 펩티드(5μM FoxM1-S25A peptide)처리에 의해 종양관련 대식세포 분화성과 종양세포 면역회피능에 억제효과가 있는 확인하기 위해서 STAT1, VEGFA, c-fos, IL6, CD274 mRNA 변화를 관찰하였다.Lastly, treatment with FoxM1 non-phosphorylated point mutant peptide (5μM FoxM1-S25A peptide) under overexpression conditions of phosphorylated point mutant of FoxM1 in A549 cells confirmed the inhibitory effect on tumor-related macrophage differentiation and tumor cell immune evasion ability. To this end, changes in STAT1, VEGFA, c-fos, IL6, and CD274 mRNA were observed.
FoxM1 인산화 점 돌연변이체의 과발현 시스템이 구축된 세포에 3ug/ml doxycycline 24시간 처리하여 과발현을 유도 후 FoxM1 비인산화 점 돌연변이 펩티드 처리를 24시간 처리하여 STAT1, VEGFA, c-fos, IL6, CD274의 mRNA 발현량을 확인하였다. 도 10G에서와 같이, 펩티드 처리에 의해서 FoxM1의 과발현에 의해서 증가된 STAT1, VEGFA, c-fos, IL6, CD274 mRNA 발현이 억제되는 것을 관찰하였다. Cells constructed with an overexpression system for FoxM1 phosphorylated point mutants were treated with 3ug/ml doxycycline for 24 hours to induce overexpression, and then treated with FoxM1 non-phosphorylated point mutant peptide for 24 hours to induce mRNA expression of STAT1, VEGFA, c-fos, IL6, and CD274. The expression level was confirmed. As shown in Figure 10G, it was observed that STAT1, VEGFA, c-fos, IL6, and CD274 mRNA expression, which was increased by overexpression of FoxM1, was suppressed by peptide treatment.
이상의 결과로, FoxM1 비인산화 점 돌연변이체 펩티드가 폐암세포에서의 전이성과 종양관련 대식세포 분화성과 종양세포 면역회피능에 억제효과가 있음을 시사하였다.The above results suggested that FoxM1 non-phosphorylated point mutant peptide had an inhibitory effect on metastatic properties of lung cancer cells, tumor-related macrophage differentiation, and tumor cell immune evasion ability.
실시예 2Example 2
<실시예 2.1> TGF-β에 의해 유도된 암전이 조건에서 FoxM1과 EMT 마커의 발현 분석<Example 2.1> Expression analysis of FoxM1 and EMT markers in cancer metastasis conditions induced by TGF-β
본 발명자는 폐암의 상피간엽이행 과정에서 FoxM1의 발현의 변화를 관찰하기 위해 비소세포폐암 세포주 A549, NCI-H358, NCI-H460을 각각 TGF-β 처리하여 EMT 마커를 관찰하였다.To observe changes in FoxM1 expression during the epithelial-mesenchymal transition of lung cancer, the present inventors treated non-small cell lung cancer cell lines A549, NCI-H358, and NCI-H460 with TGF-β, respectively, and observed EMT markers.
구체적으로 각각 5X104 개/ml 분주 후 다음날에 TGF-β를 48시간 처리하여 암전이를 유도한 후 면역블랏법을 이용하여 mesenchymal marker인 N-cadherin, SNAl1, SNAI2의 단백질 발현량이 현저히 증가하고 epithelial marker인 E-cadherin이 현저히 감소되는 것을 보고 EMT가 활성화된 것을 알 수 있었다. 또한 TGF-β 단백질이 TGF-β 계열 수용체에 결합하면 수용체의 인산화 및 SMAD 복합체 활성화로 이어져 EMT 프로그램을 활성화시킨다고 이미 밝혀져 있다 (Valcourt Ulrich, et al., Mol Biol Cell (2005) 16(4) 1987-2002). 이 시스템에서도 TGF-β를 처리시 인산화 Smad-2의 발현이 증가되는 것을 관찰할 수 있었다. 이런 조건하에서 우리는 FoxM1의 단백질 발현도 증가함을 관찰할 수 있었다 도 11). Specifically, after dispensing 5X10 4 cells/ml each, the next day, TGF-β was treated for 48 hours to induce cancer metastasis. Then, using immunoblotting, the protein expression levels of mesenchymal markers N-cadherin, SNAl1, and SNAI2 were significantly increased and epithelial It was confirmed that EMT was activated by seeing that the marker E-cadherin was significantly reduced. In addition, it has already been shown that when TGF-β protein binds to the TGF-β family receptor, it leads to phosphorylation of the receptor and activation of the SMAD complex, thereby activating the EMT program (Valcourt Ulrich, et al., Mol Biol Cell (2005) 16(4) 1987 -2002). In this system as well, it was observed that the expression of phosphorylated Smad-2 increased upon treatment with TGF-β. Under these conditions, we observed that the protein expression of FoxM1 also increased (Figure 11).
이러한 결과는 전이성 폐암에서 FoxM1의 발현이 높아진다는 것을 알 수 있었다.These results showed that FoxM1 expression was elevated in metastatic lung cancer.
<실시예 2.2> 렌티바이러스를 이용한 FoxM1 원형 발현 세포 선별 후 상피간엽이행 효과 평가<Example 2.2> Evaluation of epithelial-mesenchymal transition effect after selection of FoxM1 prototype expressing cells using lentivirus
FoxM1 단백질의 암세포 상피간엽이행 조절효과를 검토하기 위하여, 폐암세포에 FoxM1 원형 단백질을 발현시키는 렌티바이러스를 감염시키고자 다음과 같이 암세포를 배양하였다. To examine the effect of FoxM1 protein on regulating epithelial-mesenchymal transition of cancer cells, lung cancer cells were cultured as follows to infect lung cancer cells with a lentivirus expressing the FoxM1 circular protein.
폐암 세포인 A549에 FoxM1의 원형(WT)을 발현시키는 안정화된 세포주를 구축하기 위해서 먼저 pLVX-Tet3G 발현성 렌티바이러스를 감염시키고 G418을 5일 동안 500μg/ml 처리하여 감염된 세포를 선별하였다. 이렇게 선별된 A549Tet3G 세포에 FoxM1의 원형(WT)과 대조군(Mock)을 발현시키는 렌티바이러스를 감염시킨 후 puromycin을 48시간동안 2μg/ml 처리하여 안정화된 세포주를 구축하였다. 구축된 세포에 2μg/ml doxycycline 처리하여 FoxM1의 원형 발현을 유도 후 FoxM1 원형이 발현되었는지 면역블랏법을 이용하여 단백질 발현을 관찰하였다. 도 12A와 같이 FoxM1의 원형이 잘 발현되었음을 관찰할 수 있었으며, mesenchymal marker인 N-cadherin, vimentin 발현이 각각 2.2배, 2.8배 증가되었고 epithelial marker인 E-cadherin 0.6배로 발현이 감소된 것을 관찰할 수 있었다. 또한 도 12B와 같이 실시간 중합효소 연쇄반응을 이용하여 FoxM1 원형이 4.4배로 발현이 잘 되었고 mesenchymal marker인 N-cadherin, vimentin 발현이 각각 1.5배, 1.7배 증가되었고 epithelial marker인 E-cadherin 발현이 0.8배로 감소된 것을 관찰할 수 있었다. To construct a stable cell line expressing the original form (WT) of FoxM1 in A549 lung cancer cells, the cells were first infected with pLVX-Tet3G-expressing lentivirus and treated with 500 μg/ml of G418 for 5 days to select infected cells. The selected A549 Tet3G cells were infected with lentiviruses expressing the original form (WT) and control group (Mock) of FoxM1, and then treated with 2 μg/ml puromycin for 48 hours to construct a stabilized cell line. The constructed cells were treated with 2μg/ml doxycycline to induce the original expression of FoxM1, and protein expression was observed using immunoblot to determine whether the original FoxM1 was expressed. As shown in Figure 12A, it was observed that the original form of FoxM1 was well expressed, and the expression of the mesenchymal marker N-cadherin and vimentin was increased by 2.2-fold and 2.8-fold, respectively, and the expression of the epithelial marker E-cadherin was decreased by 0.6-fold. there was. In addition, as shown in Figure 12B, using real-time polymerase chain reaction, the original FoxM1 was expressed 4.4-fold, the expression of mesenchymal markers N-cadherin and vimentin increased 1.5-fold and 1.7-fold, respectively, and the expression of epithelial marker E-cadherin increased 0.8-fold. A decrease could be observed.
이상의 결과로, 폐암세포에서 FoxM1 원형의 과발현은 상피간엽이행을 촉진시키는 효과가 있음을 시사한다.The above results suggest that overexpression of the original FoxM1 in lung cancer cells has the effect of promoting epithelial-mesenchymal transition.
<실시예 2.3> FoxM1 원형 단백질에 의한 전이성 촉진 효과 평가<Example 2.3> Evaluation of metastatic promotion effect by FoxM1 circular protein
FoxM1 원형 단백질을 발현시키는 폐암세포 A549에서 암세포의 이동성에 미치는 효과를 관찰하기 위해 세포이동성 실험(migration assay)을 실시하였다. A migration assay was performed to observe the effect on cancer cell mobility in lung cancer cells A549 expressing the FoxM1 circular protein.
구체적으로, 대조군과 FoxM1 원형 단백질을 발현시키는 폐암세포 A549를 5 x 104 개의 세포를 8.0 μ￥μm, 24 well insert에 분주하고 24 well plate에 10% 혈청(FBS)이 포함된 배지를 분주한 뒤 inset를 넣어 주었다. 양성 대조군의 경우, 5 ng/ml TGF-β가 포함된 RPMI 1640(10% FBS)를 0.5 ml 분주하여 사용하였다. 세포분주 72시간 후, 4% 파라포름알데히드(paraformaldehyde) 500 ㎕를 분주하고 1XPBS로 3회 세척하여 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도(intensity)를 Odyssey infrared imaging system을 이용하여 측정하였다. 대조군의 염색된 정도(intensity)를 1이라고 할 때 양성군과 실험군에서의 상대적 염색된 정도를 산출하여 그래프를 표시하였다.Specifically, 5 I added the back inset. For the positive control, 0.5 ml of RPMI 1640 (10% FBS) containing 5 ng/ml TGF-β was used. 72 hours after cell distribution, 500 ㎕ of 4% paraformaldehyde was dispensed, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the intensity of staining was measured using the Odyssey infrared imaging system. Assuming that the staining intensity (intensity) of the control group was 1, the relative staining intensity of the positive group and the experimental group was calculated and displayed on a graph.
연구 결과, FoxM1 원형 단백질을 발현시킨 실험군에서 양성대조군인 TGF-β 처리군과 같이 향상성을 보여주며 염색강도(intensity) 수치가 대조군 대비 3배까지 증가한 것을 관찰할 수 있었다(도 12C). 이 결과로부터 FoxM1 원형 단백질은 폐암의 전이성을 촉진하는 것을 알 수 있었다. As a result of the study, it was observed that the experimental group expressing the FoxM1 circular protein showed improvement similar to the positive control group, the TGF-β treatment group, and the staining intensity value increased up to 3 times compared to the control group (Figure 12C). From these results, it was found that the FoxM1 circular protein promotes the metastasis of lung cancer.
<실시예 2.4> FoxM1 원형 단백질에 의한 침습성 촉진 효과 평가<Example 2.4> Evaluation of the effect of promoting invasiveness by FoxM1 circular protein
FoxM1 원형 단백질을 발현시키는 폐암세포 A549에서 암세포의 침습성에 미치는 효과를 관찰하기 위해 마트리겔을 이용한 침습성 실험(invasion assay)을 실시하였다. An invasion assay using Matrigel was performed to observe the effect on cancer cell invasiveness in lung cancer cell A549 expressing the FoxM1 circular protein.
구체적으로, 4℃에서 16~20시간동안 마트리겔을 완전히 녹인 후 마트리겔을 1 mg/ml이 되도록 차가운 serum free RPMI 1640(4℃)으로 희석하였다. 이를 8.0 mm 24 well 인서트(insert)에 100 μ￥μl의 마트리겔 mixture(1 mg/ml)를 넣고 37℃ 배양기에서 12-20시간 동안 굳혀주었다. 굳은 마트리겔 insert에 대조군(Mock)과 실험군 FoxM1 원형(WT) 단백질을 발현시키는 폐암세포 A549들을 1X10 cells/well의 세포수로 serum free RPMI 1640 (36℃)에 희석하여 insert에 분주하였다. 여기에 36℃의 따뜻한 RPMI 1640 (10% FBS)을 0.5 ml/well씩 분주하였다. 양성 대조군의 경우 5 ng/ml TGF-β가 포함된 36℃ RPMI 1640(10% FBS)를 0.5 ml 분주하여 사용하였다. 이 후 3일에 한 번씩 배지를 교환해주고 침습되는 정도를 관찰하였으며, 암세포의 침습이 충분히 일어난 것으로 관찰된 7일 차에 배지를 제거하고 1XPBS로 세척한 후, insert 안쪽의 cell들을 면봉으로 긁어 내었고, 1XPBS로 세척하여 insert 내부에 cell과 마트리겔의 잔여물이 남지 않도록 제거하였다. Insert 바깥 면이 있는 24 well에 4% paraformaldehyde 500ul를 분주하고 5분간 실온에서 incubation한 후 1XPBS로 3회 세척하여, 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도를 DMSO로 녹인 후 590nm에서 파장을 측정하였다. 대조군의 흡광도를 1이라고 할 때 각 실험군에서의 상대적 흡광도를 산출하여 그래프를 표시하였다.Specifically, Matrigel was completely dissolved at 4°C for 16 to 20 hours, and then Matrigel was diluted to 1 mg/ml with cold serum-free RPMI 1640 (4°C). 100 μ￥μl of Matrigel mixture (1 mg/ml) was added to an 8.0 mm 24 well insert and hardened in a 37°C incubator for 12-20 hours. Lung cancer cells A549 expressing the control (Mock) and experimental group FoxM1 prototype (WT) protein were diluted in serum free RPMI 1640 (36°C) at a cell count of 1X10 cells/well and dispensed onto the hardened Matrigel insert. Here, 0.5 ml/well of warm RPMI 1640 (10% FBS) at 36°C was dispensed. For the positive control, 0.5 ml of 36°C RPMI 1640 (10% FBS) containing 5 ng/ml TGF-β was used. After that, the medium was changed once every three days and the degree of invasion was observed. On the 7th day, when it was observed that cancer cell invasion had sufficiently occurred, the medium was removed, washed with 1XPBS, and the cells inside the insert were scraped off with a cotton swab. It was washed with 1XPBS to remove any remaining cells and matrigel inside the insert. 500ul of 4% paraformaldehyde was dispensed into 24 wells on the outer side of the insert, incubated at room temperature for 5 minutes, washed three times with 1XPBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed 5 times with 1XPBS, and the degree of staining was dissolved in DMSO and the wavelength was measured at 590 nm. Assuming that the absorbance of the control group was 1, the relative absorbance of each experimental group was calculated and displayed on a graph.
연구 결과, FoxM1 원형 단백질을 발현시킨 실험군에서 양성대조군인 TGF-beta 처리군과 같은 양상으로 흡광도 수치가 대조군 대비 근 20배까지 증가한 것을 관찰할 수 있었다(도 12D). 따라서 FoxM1 원형은 폐암세포의 침습성을 촉진하는 효과가 뛰어남을 알 수 있었다.As a result of the study, it was observed that in the experimental group expressing the FoxM1 circular protein, the absorbance value increased to nearly 20 times that of the control group in the same manner as the positive control group, the TGF-beta treatment group (Figure 12D). Therefore, it was found that the original FoxM1 was highly effective in promoting the invasiveness of lung cancer cells.
<실시예 2.5> FoxM1의 원형 단백질에 의한 대식세포의 분화능 평가<Example 2.5> Evaluation of differentiation capacity of macrophages using the original FoxM1 protein
FoxM1 원형 단백질이 발현되는 암세포에서 종양미세환경에서의 대식세포 (Macrophage cell)의 분화(polarization)에 영향을 주는지 보고자 인간 단핵구 THP-1 세포와 FoxM1 원형 단백질이 과발현된 세포를 공배양한 후, THP-1 세포에서 M1, M2 마커를 관찰하였다. FoxM1 원형 단백질이 발현되는 암세포 (4x104개/ml)과 THP-1 세포 (1.2x105개/ml)를 48h동안 공배양 후 THP-1 세포에서 M1 마커인 INOS, IL12B의 mRNA 발현은 변화가 없으나 M2 마커인 IL10, CD163, CD206, TGFB1, VEGFA의 mRNA 발현은 모두 FoxM1 원형에서 증가함을 관찰하였다(도 13A). 그중 CD206, TGFB1, VEGFA는 M2d-종양 관련 대식세포(M2d-TAM)의 마커(JAYASINGAM, Sharmilla Devi, et al., Frontiers in Oncology (2020) 9: 1512.)로 알려져 있으며, FoxM1 원형 단백질이 발현되는 A549 세포와 공배양한 THP-1 세포에서 M2d-종양 관련 대식세포의 마커들이 발현이 높게 관찰되었으므로 FoxM1 원형의 발현으로 인하여 THP-1 세포가 M2d-TAM로 분화됨을 알 수 있었다. To see whether cancer cells expressing the FoxM1 circular protein affect polarization of macrophages in the tumor microenvironment, human monocyte THP-1 cells and cells overexpressing the FoxM1 circular protein were co-cultured, and THP M1 and M2 markers were observed in -1 cells. After co-culturing cancer cells (4x10 4 cells/ml) expressing the FoxM1 circular protein and THP-1 cells (1.2x10 5 cells/ml) for 48 h, there was no change in the mRNA expression of INOS and IL12B, M1 markers, in THP-1 cells. However, the mRNA expression of M2 markers IL10, CD163, CD206, TGFB1, and VEGFA were all observed to increase in the original FoxM1 group (Figure 13A). Among them, CD206, TGFB1, and VEGFA are known as markers of M2d-tumor-associated macrophages (M2d-TAM) (JAYASINGAM, Sharmilla Devi, et al., Frontiers in Oncology (2020) 9: 1512.), and FoxM1 circular protein is expressed. High expression of markers of M2d-tumor-related macrophages was observed in THP-1 cells co-cultured with A549 cells, indicating that THP-1 cells were differentiated into M2d-TAMs due to the expression of the original FoxM1.
또한 앞서 실험한 조건으로 인간 단핵구 THP-1 세포와 FoxM1 원형 단백질이 과발현된 세포군들을 각각 48시간 공배양 후에 FoxM1 원형 단백질이 발현되는 A549 세포에서 M2d 유도 인자인 IL4, IL6, IL10, VEGFA의 mRNA 발현수준을 분석한 결과 이들이 증가하였음을 관찰할 수 있었고, 면역회피 인자인 CD274 mRNA 발현도 증가하였음을 관찰하였다(도 13B). FoxM1 원형 단백질이 발현되는 A549 세포 (4x104개/ml)을 48h 동안 배양한 배지에서 TGFB1, VEGFA 단백질의 양을 ELISA로 측정한 결과, FoxM1 원형(WT)에서 대조군(Mock) 대비 모두 현저히 증가되었음을 관찰하였다(도 13C). In addition, after 48 hours of co-cultivation of human monocyte THP-1 cells and cell groups overexpressing the FoxM1 circular protein under the conditions previously tested, mRNA expression of M2d-inducing factors IL4, IL6, IL10, and VEGFA was observed in A549 cells expressing the FoxM1 circular protein. As a result of analyzing the levels, it was observed that these increased, and the expression of CD274 mRNA, an immune evasion factor, was also observed to increase (Figure 13B). As a result of measuring the amount of TGFB1 and VEGFA proteins in the medium in which A549 cells ( 4x10 cells/ml) expressing the FoxM1 circular protein were cultured for 48 h, the amounts of TGFB1 and VEGFA proteins were measured by ELISA, and it was found that they were all significantly increased in the FoxM1 circular (WT) compared to the control (Mock). observed (Figure 13C).
또한 마우스의 폐조직에서 광범위 대식세포 마커인 CD68와 종양관련대식세포 마커인 CD163 마커를 면역염색법을 이용하여 조직에서 분석한 결과, FoxM1 원형 단백질이 과발현된 세포를 주사한 마우스 폐조직에서 CD68, CD163 마커의 발현이 모두 증가됨을 확인하였다(도 13D). 본 연구결과를 통하여 FoxM1 원형 단백질이 발현되는 폐암세포에서 M2 종양관련 대식세포의 분화를 촉진하는 효과를 관찰할 수 있었다.In addition, as a result of analyzing CD68, a broad macrophage marker, and CD163, a tumor-related macrophage marker, in mouse lung tissue using immunostaining, CD68 and CD163 were found in mouse lung tissue injected with cells overexpressing the FoxM1 circular protein. It was confirmed that the expression of all markers was increased (Figure 13D). Through the results of this study, we were able to observe the effect of promoting differentiation of M2 tumor-related macrophages in lung cancer cells expressing the FoxM1 circular protein.
<실시예 2.6> FoxM1 shRNA의 상피간엽이행 억제 효과 평가 <Example 2.6> Evaluation of the inhibitory effect of FoxM1 shRNA on epithelial-mesenchymal transition
FoxM1의 mRNA 발현 억제 효과를 확인하기 위해서, shRNA 및 이를 포함하는 렌티바이러스를 제작하였다. To confirm the effect of FoxM1 on suppressing mRNA expression, shRNA and a lentivirus containing it were produced.
구체적으로 FoxM1의 mRNA 발현을 억제하기 위하여 사람의 FoxM1 mRNA(Human FoxM1 mRNA [NM_001243088.2]) 서열 중 187-207(타겟 #1), 709-729(타겟 #2) 위치의 뉴클레오티드 서열을 각각 타겟으로 하는 shRNA를 만들고자 다음과 같이 프라이머를 제작하였다. FoxM1 mRNA 서열 중 187-207(타겟 #1) 위치의 뉴클레오티드 서열을 타겟하는 포워드 프라이머로5'- ccgg- CAT CAG AGG AGG AAC CTA AGA - ctcgag - TCT TAG GTT CCT CCT CTG ATG - tttttg - 3' 사용하였고, 리버스 프라이머로 5' - aattcaaaaa - CAT CAG AGG AGG AAC CTA AGA - ctcgag - TCT TAG GTT CCT CCT CTG ATG - 3'를 사용하였다. 또한 FoxM1 mRNA 서열 중 709-729(타겟 #2) 위치의 뉴클레오티드 서열을 타겟하는 포워드 프라이머로 5' - ccgg - AGC AAG AGA TGG AGG AAA AGG- ctcgag - CCT TTT CCT CCA TCT CTT GCT - tttttg - 3'를 사용하였고, 리버스 프라이머로 5' - aattcaaaaa - AGC AAG AGA TGG AGG AAA AGG - ctcgag - CCT TTT CCT CCA TCT CTT GCT - 3'를 사용하였다. 이를 기반으로 pLKO-puro.1 벡터를 이용한 pLKO-puro.1-shFoxM1 플라스미드를 제작하였다. 이를 pHR'-CMV-VSVG, pHR'-CMV-deltaR8.2와 함께 HEK293 세포 형질감염을 통해 발현시킨 후, 세포의 배양배지를 모아 렌티바이러스를 생산하였다. 원심분리기를 이용하여 상기 렌티바이러스를 농축시켰다. 바이러스 발현 확인을 위하여 A549 세포를 5X104 개/ml으로 배양한 후, 다음 날 감염 버퍼(Infection Buffer; 10mM HEPES, 1 mg/ml 폴리브렌에 렌티바이러스를 20 μ￥μ/well로 첨가하여 암세포를 감염시켰다. 24시간이 지난 후 puromycin를 2 μg/ml로 48시간동안 처리하여 shFoxM1 감염된 세포를 선별하였다. Specifically, to suppress the mRNA expression of FoxM1, target the nucleotide sequences at positions 187-207 (target #1) and 709-729 (target #2) of the human FoxM1 mRNA (Human FoxM1 mRNA [NM_001243088.2]) sequence. To create shRNA, primers were prepared as follows. Use 5'- ccgg- CAT CAG AGG AGG AAC CTA AGA - ctcgag - TCT TAG GTT CCT CCT CTG ATG - tttttg - 3' as a forward primer targeting the nucleotide sequence at positions 187-207 (target #1) of the FoxM1 mRNA sequence. 5' - aattcaaaaa - CAT CAG AGG AGG AAC CTA AGA - ctcgag - TCT TAG GTT CCT CCT CTG ATG - 3' was used as the reverse primer. In addition, the forward primer targeting the nucleotide sequence at position 709-729 (target #2) of the FoxM1 mRNA sequence is 5' - ccgg - AGC AAG AGA TGG AGG AAA AGG- ctcgag - CCT TTT CCT CCA TCT CTT GCT - tttttg - 3' was used, and 5' - aattcaaaaa - AGC AAG AGA TGG AGG AAA AGG - ctcgag - CCT TTT CCT CCA TCT CTT GCT - 3' was used as the reverse primer. Based on this, pLKO-puro.1-shFoxM1 plasmid was created using the pLKO-puro.1 vector. This was expressed through HEK293 cell transfection along with pHR'-CMV-VSVG and pHR'-CMV-deltaR8.2, and the culture medium of the cells was collected to produce lentivirus. The lentivirus was concentrated using a centrifuge. To confirm virus expression, A549 cells were cultured at 5 After 24 hours of infection, shFoxM1-infected cells were selected by treating them with 2 μg/ml puromycin for 48 hours.
먼저 본 발명자는 제작한 FoxM1 shRNA에 의한 FoxM1 발현억제 효과를 확인하고자 puromycine 2 μg/ml로 48시간동안 처리를 통하여 선별된 각각의 FoxM1 shRNA에 감염된 세포에서 FoxM1의 mRNA 발현과 단백질 발현정도를 확인하였다.First, to confirm the effect of suppressing FoxM1 expression by the produced FoxM1 shRNA, the present inventor confirmed the mRNA and protein expression levels of FoxM1 in cells infected with each selected FoxM1 shRNA by treatment with puromycine 2 μg/ml for 48 hours. .
도 14A, 14B에 나타낸 바와 같이 각각의 FoxM1 shRNA(shFoxM1)에 감염된 폐암세포 A549는 대조군 shRNA(shCtrl)에 감염된 세포에 비하여 FoxM1의 mRNA의 발현이 감소하였고, 이는 FoxM1의 발현이 억제되었음을 관찰하였다. 특히 FoxM1 mRNA 서열에서 709-729(타겟 #2) 위치의 뉴클레오티드 서열을 타겟으로 하는 shRNA가 FoxM1의 발현억제 효과가 가장 좋은 것으로 관찰되었다. As shown in Figures 14A and 14B, the expression of FoxM1 mRNA was decreased in lung cancer cells A549 infected with each FoxM1 shRNA (shFoxM1) compared to cells infected with control shRNA (shCtrl), which showed that FoxM1 expression was suppressed. In particular, shRNA targeting the nucleotide sequence at positions 709-729 (target #2) in the FoxM1 mRNA sequence was observed to have the best effect in suppressing FoxM1 expression.
따라서 본 발명자는 하기에 FoxM1 발현억제 연구 시에 FoxM1 mRNA 서열에서 709-729(타겟 #2) 위치의 뉴클레오티드 서열을 타겟하는 shRNA를 사용하였다.Therefore, the present inventor used shRNA targeting the nucleotide sequence at position 709-729 (target #2) in the FoxM1 mRNA sequence when studying the inhibition of FoxM1 expression below.
본 발명자는 FoxM1 shRNA 처리로 FoxM1의 발현 억제가 폐암세포 이동성을 억제하는지 알기 위해 세포이동성실험(migration assay)를 진행하였다. 구체적으로 폐암세포 A549에 각각 대조군 shRNA(shCtrl)와 FoxM1 shRNA(shFoxM1) 바이러스를 감염시켰다. 다음날, 2μ￥μg/ml 퓨로마이신(puromycin)을 48시간동안 처리 후 선별된 shFoxM1이 발현되는 세포를 6 well plate에 2x105 cells/ml로 분주하였으며 24시간 후 pipette tip를 사용하여 일정한 간격으로 스크래치를 내었다. 스크래치를 낸 후 24시간 간격으로 세포의 간격과 이동성을 현미경으로 관찰하고 현미경에서의 세포 촬영을 통하여 간격이 복구되는 정도를 세포사이의 거리로 측정하였다. 측정된 수치를 대조군의 거리를 100%라고 할 때 상대적 이동 거리를 산출한 후, %로 그래프를 표시하였다 (도 14C).The present inventors conducted a cell migration assay to determine whether inhibition of FoxM1 expression by FoxM1 shRNA treatment inhibits lung cancer cell mobility. Specifically, lung cancer cells A549 were infected with control shRNA (shCtrl) and FoxM1 shRNA (shFoxM1) viruses, respectively. The next day, after treatment with 2μ￥μg/ml puromycin for 48 hours, the selected cells expressing shFoxM1 were distributed at 2x105 cells/ml in a 6-well plate, and after 24 hours, scratched at regular intervals using a pipette tip. issued. After scratching, the spacing and mobility of cells were observed under a microscope at intervals of 24 hours, and the extent to which the spacing was restored was measured as the distance between cells by photographing the cells under the microscope. When the measured value was assumed to be 100% of the distance of the control group, the relative moving distance was calculated and then graphed as a percentage (Figure 14C).
상대적 이동 거리 (%) = 실험군에서의 측정수치 x 100 / 대조군의 측정수치Relative moving distance (%) = measured value in the experimental group x 100 / measured value in the control group
연구결과, FoxM1 shRNA 처리에 의한 FoxM1 발현억제로 폐암세포 자체의 전이성을 감소시켰는데 특히 72시간에는 대조군의 이동성이 0%라고 설정할 때 약 -20% 이상의 이동성을 감소시켰다(도 14D). 따라서 FoxM1 shRNA에 의한 FoxM1 발현 억제는 암세포 자체의 전이성에 대한 강력한 억제효과가 있음을 알 수 있었다. As a result of the study, inhibition of FoxM1 expression by FoxM1 shRNA treatment reduced the metastatic properties of the lung cancer cells themselves. In particular, at 72 hours, when the mobility of the control group was set to 0%, the mobility was reduced by more than -20% (Figure 14D). Therefore, it was found that inhibition of FoxM1 expression by FoxM1 shRNA had a strong inhibitory effect on the metastasis of cancer cells themselves.
본 발명자는 TGF-β를 처리에 의해 유도된 암전이가 FoxM1 shRNA 처리로 FoxM1 mRNA 발현 억제가 상피간엽이행 마커 및 관련 인자들의 mRNA 발현 및 단백질 발현 변화 관찰하였다. The present inventors observed that cancer metastasis induced by treatment with TGF-β suppressed FoxM1 mRNA expression by FoxM1 shRNA treatment and changed the mRNA and protein expressions of epithelial-mesenchymal transition markers and related factors.
구축된 FoxM1을 억제하는 세포주에 5ng/ml TGF-β를 48시간 동안 처리하여 암전이를 유도한 조건에서 실시간 중합효소 연쇄반응(Real-time PCR)을 통해 상피간엽이행 관련 인자들인 CDH1, CDH2 발현을 관찰하였다(도 14E).Expression of epithelial-mesenchymal transition-related factors CDH1 and CDH2 through real-time polymerase chain reaction (Real-time PCR) under conditions in which cancer metastasis was induced by treating the constructed FoxM1-inhibiting cell line with 5ng/ml TGF-β for 48 hours. was observed (Figure 14E).
FoxM1 shRNA(shFoxM1)에 감염된 폐암세포 A549는 대조군 shRNA(shCtrl)에 감염된 세포에 비하여 FoxM1의 mRNA의 발현이 감소하였고, 이는 FoxM1의 발현이 억제되었으며, 암전이 환경을 유도하는 TGF-β를 처리한 조건에서도 FoxM1의 발현이 억제되었음을 관찰하였다(도 14E). TGF-β 처리에 의한 상피 마커인 CDH1 발현의 감소가 shFoxM1 처리에 의한 FoxM1의 발현 억제로 CDH1 발현의 감소가 억제되었으며, 또한 TGF-β 처리에 의한 중간엽 마커인 CDH2 발현의 증가가 FoxM1의 발현 억제를 의해 억제되었음을 관찰하였다(도 14E).Lung cancer cells A549 infected with FoxM1 shRNA (shFoxM1) showed decreased expression of FoxM1 mRNA compared to cells infected with control shRNA (shCtrl), which inhibited the expression of FoxM1 and treated with TGF-β, which induces a cancer metastatic environment. It was observed that the expression of FoxM1 was suppressed under these conditions (Figure 14E). The decrease in CDH1 expression, an epithelial marker, by TGF-β treatment was suppressed by suppressing the expression of FoxM1 by shFoxM1 treatment, and the increase in CDH2 expression, a mesenchymal marker, by TGF-β treatment was also suppressed by the expression of FoxM1. It was observed that it was suppressed by inhibition (Figure 14E).
<실시예 2.7> FoxM1 shRNA 처리에 의한 세포 사멸 유도 효과 <Example 2.7> Cell death induction effect by FoxM1 shRNA treatment
shFoxM1 처리에 의한 FoxM1의 발현 억제로 A549 세포에서 암세포의 세포사멸정도를 관찰하기 위하여 caspase-3 assay을 진행하여 caspase-3 효소의 활성을 측정하였다(도 14F). A549 세포에 shCtrl, shFoxM1#187, shFoxM1#709를 발현하는 바이러스를 48시간 동안 감염 시킨 후 puromycin 선별 과정 48시간 진행하고 세포를 얻은 후 lysis하였다. caspase-3의 형광기질(Ac-DEVD-AMC)와 1시간 반응 후 전자분광분석기(spectramax M4 system)을 이용하여 형광값(Ex. 380nm/Emi. 430nm)을 측정하였다. 측정된 형광값은 caspase-3 활성으로 상대 형광 단위(relative fluorescence units, RFU)로 표시하여 세포사멸정도를, 대조군의 측정값을 1이라고 할 때 각 실험군에서의 상대적 측정값의 정도를 산출하여 그래프로 표시하였다. 그 결과, 대조군 shRNA(shCtrl) 처리군 세포 대비, shFoxM1#709 세포에서 caspase-3 효소의 활성이 5배 이상 증가됨을 관찰하였으며, shFoxM1#187 세포에서 caspase-3 효소의 활성이 2배 이상 증가됨을 관찰하였다. 따라서, shFoxM1 처리에 의한 FoxM1의 발현 억제로 세포사멸성을 유도 및 증가시키는 것을 알 수 있었다.In order to observe the degree of apoptosis of cancer cells in A549 cells by suppressing the expression of FoxM1 by shFoxM1 treatment, caspase-3 assay was performed to measure the activity of caspase-3 enzyme (Figure 14F). A549 cells were infected with viruses expressing shCtrl, shFoxM1#187, and shFoxM1#709 for 48 hours, followed by puromycin selection for 48 hours, and cells were obtained and lysed. After reacting with the fluorescent substrate (Ac-DEVD-AMC) of caspase-3 for 1 hour, the fluorescence value (Ex. 380nm/Emi. 430nm) was measured using an electron spectrometer (spectramax M4 system). The measured fluorescence value is expressed as relative fluorescence units (RFU) for caspase-3 activity, indicating the degree of cell death. When the measured value of the control group is 1, the degree of relative measured value in each experimental group is calculated and graphed. It is indicated as . As a result, compared to control shRNA (shCtrl) treated group cells, it was observed that the activity of caspase-3 enzyme was increased more than 5-fold in shFoxM1#709 cells, and that the activity of caspase-3 enzyme was increased more than 2-fold in shFoxM1#187 cells. observed. Therefore, it was found that inhibition of FoxM1 expression by shFoxM1 treatment induced and increased apoptosis.
<실시예 2.8> FoxM1의 인산화 점 돌연변이에서 FoxM1 shRNA에 의한 대식세포의 분화능 평가<Example 2.8> Evaluation of differentiation ability of macrophages by FoxM1 shRNA in phosphorylation point mutant of FoxM1
본 발명자는 FoxM1 shRNA 처리로 FoxM1의 발현 억제가 FoxM1의 인산화 점 돌연변이에서 FoxM1 발현이 감소하는지 관찰하였다. The present inventors observed whether inhibition of FoxM1 expression by FoxM1 shRNA treatment resulted in a decrease in FoxM1 expression in phosphorylation point mutants of FoxM1.
구체적으로 폐암세포 A549에 FoxM1 인산화 점 돌연변이(S25E)가 발현되는 세포에 각각 대조군 shRNA(shCtrl)와 FoxM1 shRNA(shFoxM1) 바이러스를 감염시켰다. 감염시킨 세포를 실시간 중합효소 연쇄반응(Real-time PCR)을 통해 FoxM1의 mRNA 발현을 관찰한 결과 FoxM1 shRNA 처리로 FoxM1의 발현 억제가 FoxM1의 인산화 점 돌연변이에서 감소하는 것을 관찰할 수 있었다(도 15A). Specifically, lung cancer cells A549 expressing the FoxM1 phosphorylation point mutation (S25E) were infected with control shRNA (shCtrl) and FoxM1 shRNA (shFoxM1) viruses, respectively. As a result of observing the mRNA expression of FoxM1 in the infected cells through real-time polymerase chain reaction (Real-time PCR), it was observed that the inhibition of FoxM1 expression by treatment with FoxM1 shRNA was reduced in the phosphorylation point mutant of FoxM1 (Figure 15A ).
그리고 상위 FoxM1 shRNA 처리를 한 암세포(4x104개/ml)와 인간 단핵구 THP-1 세포 (1.2x105개/ml) 세포를 각각 48시간 공배양 후에 THP-1에서 M2 마커 인자인 CD163, CD206, VEGFA의 mRNA 발현수준을 분석한 결과 이들이 FoxM1 인산화 점 돌연변이(S25E)가 발현되는 세포에 FoxM1 shRNA처리한 세포군에서 현저히 감소됨을 관찰할 수 있었다(도 15B). And after co-culturing cancer cells (4x10 4 cells/ml) and human monocyte THP-1 cells (1.2x10 5 cells/ml) treated with high-level FoxM1 shRNA for 48 hours, the M2 marker factors CD163, CD206, As a result of analyzing the mRNA expression level of VEGFA, it was observed that it was significantly reduced in the cell group in which FoxM1 shRNA was treated with cells expressing the FoxM1 phosphorylation point mutation (S25E) (FIG. 15B).
또한 폐암세포 A549에 FoxM1 인산화 점 돌연변이(S25E)가 발현되는 세포에 각각 대조군 shRNA(shCtrl)와 FoxM1 shRNA(shFoxM1) 바이러스를 감염시킨 세포에서 실시간 중합효소 연쇄반응(Real-time PCR)을 통하여 IFITM1의 mRNA 발현을 보았다. 결과 FoxM1의 인산화 점 돌연변이에서 FoxM1 shRNA 처리로 FoxM1의 발현 억제를 한 결과 IFITM1의 mRNA 발현이 현저히 감소하는 것을 관찰할 수 있었다(도 15C).In addition, lung cancer cell A549 expressing the FoxM1 phosphorylation point mutation (S25E) was infected with control shRNA (shCtrl) and FoxM1 shRNA (shFoxM1) viruses, respectively, through real-time polymerase chain reaction (Real-time PCR) of IFITM1. We looked at mRNA expression. As a result, in the phosphorylated point mutant of FoxM1, the expression of FoxM1 was suppressed by FoxM1 shRNA treatment, and a significant decrease in IFITM1 mRNA expression was observed (Figure 15C).
<실시예 2.9> FoxM1 억제제인 Thiostrepton 처리에 의한 FoxM1의 인산화 점 돌연변이 과발현 세포에서 상피간엽이행 및 대식세포의 분화능 억제 효과 평가<Example 2.9> Evaluation of the effect of suppressing epithelial-mesenchymal transition and differentiation of macrophages in cells overexpressing phosphorylation point mutations of FoxM1 by treatment with Thiostrepton, a FoxM1 inhibitor
본 발명자는 FoxM1 억제제인 Thiostrepton 처리로 FoxM1의 발현 억제가 FoxM1의 인산화 점 돌연변이 단백질이 과발현 된 세포에서 상피간엽이행 및 대식세포의 분화능이 억제하는지 관찰하였다. The present inventors observed whether inhibition of FoxM1 expression by treatment with Thiostrepton, a FoxM1 inhibitor, inhibits epithelial-mesenchymal transition and differentiation ability of macrophages in cells overexpressing a phosphorylated point mutant protein of FoxM1.
구체적으로 폐암세포 A549에 대조군(Mock)과 FoxM1 인산화 점 돌연변이(S25E)가 발현되는 세포에 FoxM1 억제제인 Thiostrepton를 5 μM 농도로 48h 처리하여 실시간 중합효소 연쇄반응(Real-time PCR)을 통해 상피간엽이행 관련 인자 mesenchymal 마커인 CDH2, vimentin, SNAI1, SNAI2의 발현이 FoxM1 억제제인 Thiostrepton처리 군에서 현저히 감소되는 것을 관찰할 수 있었다(도 16A).Specifically, lung cancer cell A549 control (Mock) and cells expressing FoxM1 phosphorylation point mutation (S25E) were treated with Thiostrepton, a FoxM1 inhibitor, at a concentration of 5 μM for 48 h, and epithelial-mesenchymal growth was achieved through real-time polymerase chain reaction (Real-time PCR). Expression of migration-related mesenchymal markers CDH2, vimentin, SNAI1, and SNAI2 was observed to be significantly reduced in the group treated with Thiostrepton, a FoxM1 inhibitor (Figure 16A).
또한 상위 실험조건과 같은 조건에서 실시간 중합효소 연쇄반응(Real-time PCR)을 통해 M2 유도 인자인 IL6, VEGFA와 면역관문회피 인자 CD274(PD-L1)의 발현이 FoxM1 억제제인 Thiostrepton처리 군에서도 현저히 감소되는 것을 관찰할 수 있었다(도 16B).In addition, under the same conditions as the upper experimental conditions, the expression of M2-inducing factors IL6, VEGFA, and immune checkpoint evasion factor CD274 (PD-L1) was significantly increased in the group treated with Thiostrepton, a FoxM1 inhibitor, through real-time PCR. A decrease could be observed (Figure 16B).
상기 결과로부터 본 연구에서 FoxM1 억제제인 Thiostrepton 처리에 의한 FoxM1의 인산화 점 돌연변이 과발현 세포에서 상피간엽이행 및 대식세포의 분화능과 암세포의 면역관문회피능이 억제됨을 알 수 있다. From the above results, it can be seen that in this study, epithelial-mesenchymal transition and differentiation ability of macrophages and immune checkpoint evasion ability of cancer cells were suppressed in cells overexpressing phosphorylation point mutants of FoxM1 by treatment with Thiostrepton, a FoxM1 inhibitor.

Claims (36)

  1. 서열번호 1의 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된 폴리펩티드를 포함하는, 암 치료용 약학 조성물. A pharmaceutical composition for treating cancer, comprising a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with a non-phosphorylated amino acid.
  2. 제1항에 있어서, 상기 비인산화 아미노산은 Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His, 또는 Lys인, 약학 조성물. The pharmaceutical composition of claim 1, wherein the non-phosphorylated amino acid is Gly, Ala, Val, Ile, Leu, Met, Phe, Trp, Asn, Gln, Cys, Pro, Arg, His, or Lys.
  3. 제1항에 있어서, 상기 암은 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 또는 뇌하수체 선종인, 약학 조성물. The method of claim 1, wherein the cancer is bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, and uterine cancer. , ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spine A pharmaceutical composition that is a tumor, glioma, meningioma or pituitary adenoma.
  4. 제1항에 있어서, 상기 약학 조성물은 암세포의 사멸을 유도하거나, 암세포의 성장성, 이동성, 침습성, 및 전이성으로 구성된 군으로부터 선택된 하나 이상을 억제하는, 약학 조성물. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition induces death of cancer cells or inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
  5. 서열번호 1의 24번째 아미노산 Pro 내지 27번째 아미노산 Thr을 포함하고, 연속된 10개 이상의 아미노산을 포함하며, 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된, 폴리펩티드를 포함하는, 폴리펩티드. A polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, 10 or more consecutive amino acids, and a polypeptide in which the 25th amino acid Ser is substituted with a non-phosphorylated amino acid.
  6. 제5항에 있어서, 상기 폴리펩티드는 서열번호 2, 4, 또는 5로 표시되는 아미노산 서열을 포함하는 폴리펩티드. The polypeptide according to claim 5, wherein the polypeptide comprises an amino acid sequence represented by SEQ ID NO: 2, 4, or 5.
  7. 서열번호 1의 24번째 아미노산 Pro 내지 27번째 아미노산 Thr을 포함하고, 연속된 10개 이상의 아미노산을 포함하며, 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된 폴리펩티드를 포함하는, 암 치료용 약학 조성물. A pharmaceutical composition for treating cancer, comprising a polypeptide comprising the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, 10 or more consecutive amino acids, and the 25th amino acid Ser substituted with a non-phosphorylated amino acid.
  8. 제7항에 있어서, 상기 폴리펩티드는 서열번호 2, 4, 또는 5로 표시되는 아미노산 서열을 포함하는 약학 조성물. The pharmaceutical composition according to claim 7, wherein the polypeptide comprises an amino acid sequence represented by SEQ ID NO: 2, 4, or 5.
  9. 제7항에 있어서, 상기 암은 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 또는 뇌하수체 선종인, 약학 조성물.The method of claim 7, wherein the cancer is bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, and uterine cancer. , ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spine A pharmaceutical composition that is a tumor, glioma, meningioma or pituitary adenoma.
  10. 제7항에 있어서, 상기 약학 조성물은 암세포의 사멸을 유도하거나, 암세포의 성장성, 이동성, 침습성, 및 전이성으로 구성된 군으로부터 선택된 하나 이상을 억제하는, 약학 조성물. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition induces death of cancer cells or inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
  11. 서열번호 1의 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된 폴리펩티드, 또는 제5항 또는 제6항의 폴리펩티드를 코딩하는 핵산 분자. A polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is substituted with a non-phosphorylated amino acid, or a nucleic acid molecule encoding the polypeptide of claim 5 or 6.
  12. 제11항에 있어서, 상기 핵산 분자는 The method of claim 11, wherein the nucleic acid molecule is
    서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환된 핵산 분자; Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. substituted nucleic acid molecules;
    서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환되고, 61번째 핵산 내지 90번째 핵산 서열을 포함하는 핵산 분자; Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. a nucleic acid molecule that is substituted and comprises the 61st to 90th nucleic acid sequences;
    서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환되고, 70번째 핵산 내지 99번째 핵산 서열을 포함하는 핵산 분자;Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. a nucleic acid molecule that is substituted and comprises the 70th to 99th nucleic acid sequences;
    서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환되고, 52번째 핵산 내지 81번째 핵산 서열을 포함하는 핵산 분자인, 핵산 분자. Among the nucleic acid sequences represented by SEQ ID NO: 3, the 73rd to 75th nucleic acids are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3'. A nucleic acid molecule that is substituted and comprises nucleic acid sequences 52 to 81.
  13. 제11항의 핵산 분자를 포함하는 재조합 벡터. A recombinant vector containing the nucleic acid molecule of claim 11.
  14. 제13항의 재조합 벡터를 포함하는 재조합 세포주. A recombinant cell line containing the recombinant vector of claim 13.
  15. 대상체의 암세포에서 서열번호 1로 표시되는 FoxM1 단백질 중 25번째 아미노산 Ser이 인산화되었거나 Asp 또는 Glu으로 치환된 경우, 암의 전이 가능성이 높은 것으로 판단하는 단계를 포함하는, 암의 전이 위험성 판단을 위한 정보제공방법. Information for determining the risk of cancer metastasis, including the step of determining that there is a high possibility of cancer metastasis when the 25th amino acid Ser of the FoxM1 protein represented by SEQ ID NO. 1 in the subject's cancer cells is phosphorylated or substituted with Asp or Glu. How to provide.
  16. 대상체의 암세포에서 서열번호 3으로 표시되는 핵산 서열 중 73번째 핵산 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환된 경우, 암의 전이 가능성이 높은 것으로 판단하는 단계를 포함하는, 암의 전이 위험성 판단을 위한 정보제공방법.In the subject's cancer cells, the 73rd to 75th nucleic acids in the nucleic acid sequence shown in SEQ ID NO: 3 are 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG. A method of providing information for determining the risk of cancer metastasis, including the step of determining that the possibility of cancer metastasis is high when substituted with -3'.
  17. 서열번호 1의 25번째 아미노산 Ser이 Asp, 또는 Glu로 치환된 폴리펩티드를 발현하는, 재조합 전이성 암 세포. Recombinant metastatic cancer cells expressing a polypeptide in which the 25th amino acid Ser of SEQ ID NO: 1 is replaced with Asp or Glu.
  18. 서열번호 3으로 표시되는 핵산 서열 중 73번째 내지 75번째 핵산이 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', 또는 5'-GAG-3'로 치환된 핵산을 포함하는, 재조합 전이성 암세포.Nucleic acid positions 73 to 75 of the nucleic acid sequence shown in SEQ ID NO: 3 are substituted with 5'-GAT-3', 5'-GAC-3', 5'-GAA-3', or 5'-GAG-3' Recombinant metastatic cancer cells containing nucleic acids.
  19. 서열번호 1의 24번째 아미노산 Pro 내지 27번째 아미노산 Thr을 포함하고, 연속된 10개 이상의 아미노산을 포함하며, 25번째 아미노산 Ser이 비인산화 아미노산으로 치환된 폴리펩티드를 암 치료가 필요한 대상체에게 투여하는 단계를 포함하는, 암 치료 방법. Administering a polypeptide containing the 24th amino acid Pro to the 27th amino acid Thr of SEQ ID NO: 1, containing 10 or more consecutive amino acids, and in which the 25th amino acid Ser is substituted with a non-phosphorylated amino acid to a subject in need of cancer treatment. Including, a cancer treatment method.
  20. 제19항에 있어서, 상기 폴리펩티드는 서열번호 2, 4, 또는 5로 표시되는 아미노산 서열을 포함하는, 암 치료 방법. The method of claim 19, wherein the polypeptide comprises an amino acid sequence represented by SEQ ID NO: 2, 4, or 5.
  21. 제19항에 있어서, 상기 암은 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 또는 뇌하수체 선종인, 암 치료 방법.The method of claim 19, wherein the cancer is bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, and uterine cancer. , ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spine A method of treating cancer, which is a tumor, glioma, meningioma, or pituitary adenoma.
  22. 제19항에 있어서, 상기 약학 조성물은 암세포의 사멸을 유도하거나, 암세포의 성장성, 이동성, 침습성, 및 전이성으로 구성된 군으로부터 선택된 하나 이상을 억제하는, 암 치료 방법. The cancer treatment method according to claim 19, wherein the pharmaceutical composition induces death of cancer cells or inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
  23. FOXM1 단백질을 코딩하는 유전자 또는 mRNA에 상보적으로 결합하여 FOXM1 단백질의 발현을 저하시키는 핵산분자를 포함하는, 암 치료용 약학 조성물.A pharmaceutical composition for treating cancer, comprising a nucleic acid molecule that reduces expression of the FOXM1 protein by binding complementary to the gene or mRNA encoding the FOXM1 protein.
  24. 제23항에 있어서, 상기 FOXM1 단백질을 코딩하는 유전자 또는 mRNA는 서열번호 6으로 표시되는 핵산 서열을 포함하는, 약학 조성물. The pharmaceutical composition according to claim 23, wherein the gene or mRNA encoding the FOXM1 protein comprises a nucleic acid sequence represented by SEQ ID NO: 6.
  25. 제23항에 있어서, 상기 핵산분자는 서열번호 6의 187 내지 207번째 핵산 또는 709 내지 729번째 핵산에 특이적으로 결합하는 핵산 서열을 포함하는, 약학 조성물. The pharmaceutical composition according to claim 23, wherein the nucleic acid molecule comprises a nucleic acid sequence that specifically binds to nucleic acids 187 to 207 or nucleic acids 709 to 729 of SEQ ID NO: 6.
  26. 제23항에 있어서, 상기 핵산분자는 서열번호 7 내지 10 중 어느 하나의 핵산 서열을 포함하는, 약학 조성물. The pharmaceutical composition of claim 23, wherein the nucleic acid molecule comprises a nucleic acid sequence of any one of SEQ ID NOs: 7 to 10.
  27. 제23항에 있어서, 상기 핵산분자는 shRNA, siRNA, 안티센스 RNA, 안티센스 DNA, 키메라 안티센스 DNA/RNA, miRNA, 및 라이보자임으로 구성된 군으로부터 선택된 어느 하나인, 약학 조성물. The pharmaceutical composition of claim 23, wherein the nucleic acid molecule is any one selected from the group consisting of shRNA, siRNA, antisense RNA, antisense DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme.
  28. 제23항에 있어서, 상기 암은 FOXM1 단백질을 과발현하거나, 25번째 아미노산 Ser이 Asp, 또는 Glu로 치환된 FOXM1 단백질을 발현하는 암인, 약학 조성물. The pharmaceutical composition according to claim 23, wherein the cancer is a cancer that overexpresses FOXM1 protein or expresses FOXM1 protein in which the 25th amino acid Ser is substituted with Asp or Glu.
  29. 제23항에 있어서, 상기 암은 골암, 혈액암, 폐암, 소세포폐암, 비소세포폐암, 편평상피세포암, 선암, 대세포폐암, 간암, 췌장암, 피부암, 두경부암, 피부 또는 안내 흑색종, 자궁암, 난소암, 직장암, 항문 부위의 암, 위암, 대장암, 유방암, 전립선암, 자궁암, 자궁내막암, 육종암, 갈색세포종 부신 종양, 고환생식세포종양, 자궁경부암, 성 및 생식 기관의 암종, 호지킨 질환, 식도암, 소장암, 내분비계 암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 방광암, 신장암, 신장 세포 암종, 신우 암종, 중추 신경계 (CNS)의 신생물, 신경외배엽 암, 척추 종양, 신경교종, 수막종 또는 뇌하수체 선종인, 약학 조성물.The method of claim 23, wherein the cancer is bone cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell cancer, adenocarcinoma, large cell lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, and uterine cancer. , ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, endometrial cancer, sarcoma cancer, pheochromocytoma, adrenal tumor, testicular germ cell tumor, cervical cancer, carcinoma of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spine A pharmaceutical composition that is a tumor, glioma, meningioma or pituitary adenoma.
  30. 제23항에 있어서, 상기 약학 조성물은 암세포의 사멸을 유도하거나, 암세포의 성장성, 이동성, 침습성, 및 전이성으로 구성된 군으로부터 선택된 하나 이상을 억제하는, 약학 조성물. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition induces death of cancer cells or inhibits one or more selected from the group consisting of growth, mobility, invasiveness, and metastasis of cancer cells.
  31. 서열번호 7 내지 10 중 어느 하나의 핵산 서열을 포함하고, shRNA, siRNA, 안티센스 RNA, 안티센스 DNA, 키메라 안티센스 DNA/RNA, miRNA, 및 라이보자임으로 구성된 군으로부터 선택된 어느 하나인, 핵산 분자. A nucleic acid molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 7 to 10, and being any one selected from the group consisting of shRNA, siRNA, antisense RNA, antisense DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme.
  32. 제31항에 있어서, 상기 핵산 분자는 FOXM1 단백질을 코딩하는 유전자 또는 mRNA에 상보적으로 결합하는, 핵산 분자. 32. The nucleic acid molecule of claim 31, wherein the nucleic acid molecule binds complementary to a gene or mRNA encoding a FOXM1 protein.
  33. 제31항에 있어서, 서열번호 1의 187 내지 207번째 핵산 또는 709 내지 729번째 핵산에 특이적으로 결합하는, 핵산 분자. The nucleic acid molecule according to claim 31, which specifically binds to nucleic acids 187 to 207 or nucleic acids 709 to 729 of SEQ ID NO: 1.
  34. 서열번호 7 내지 10 중 어느 하나의 핵산 서열을 포함하는 재조합 바이러스 벡터. A recombinant viral vector comprising the nucleic acid sequence of any one of SEQ ID NOs: 7 to 10.
  35. 제34항에 있어서, shRNA, siRNA, 안티센스 RNA, 안티센스 DNA, 키메라 안티센스 DNA/RNA, miRNA, 및 라이보자임으로 구성된 군으로부터 선택된 어느 하나를 발현하는, 재조합 바이러스 벡터.The recombinant viral vector according to claim 34, which expresses any one selected from the group consisting of shRNA, siRNA, antisense RNA, antisense DNA, chimeric antisense DNA/RNA, miRNA, and ribozyme.
  36. 제23항 내지 제30항 중 어느 한 항의 약학 조성물; 제31항 내지 제33항 중 어느 한 항의 핵산 분자; 또는 제34항 또는 제35항의 재조합 바이러스 벡터를 암 치료가 필요한 대상체에게 투여하는 단계를 포함하는, 암 치료 방법.The pharmaceutical composition of any one of claims 23 to 30; The nucleic acid molecule of any one of claims 31 to 33; Or a cancer treatment method comprising administering the recombinant viral vector of claim 34 or 35 to a subject in need of cancer treatment.
PCT/KR2023/014663 2022-11-16 2023-09-25 Pharmaceutical composition for treating cancer comprising foxm1 mutant or foxm1 shrna WO2024106737A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006009575A1 (en) * 2004-06-22 2006-01-26 The Board Of Trustees Of The University Of Illinois METHODS OF INHIBITING TUMOR CELL PROLIFERATION WITH FOXM1 siRNA
US7655402B2 (en) * 2004-02-06 2010-02-02 Wyeth Llc Diagnoses and therapeutics for cancer
KR20160045063A (en) * 2013-08-19 2016-04-26 에프. 호프만-라 로슈 아게 Screening method
KR20180044208A (en) * 2016-10-21 2018-05-02 서울대학교병원 Composition and method for predicting prognosis of breast cancer
KR20180056778A (en) * 2015-10-08 2018-05-29 온코세라피 사이언스 가부시키가이샤 FOXM1-derived peptides and vaccines comprising them

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7655402B2 (en) * 2004-02-06 2010-02-02 Wyeth Llc Diagnoses and therapeutics for cancer
WO2006009575A1 (en) * 2004-06-22 2006-01-26 The Board Of Trustees Of The University Of Illinois METHODS OF INHIBITING TUMOR CELL PROLIFERATION WITH FOXM1 siRNA
KR20160045063A (en) * 2013-08-19 2016-04-26 에프. 호프만-라 로슈 아게 Screening method
KR20180056778A (en) * 2015-10-08 2018-05-29 온코세라피 사이언스 가부시키가이샤 FOXM1-derived peptides and vaccines comprising them
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